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Thermo Scientific Precision water baths are designed to save valuable benchtop space with smaller footprints compared to previous models while maintaining excellent temperature uniformity and stability. With rugged construction and advanced temperature control, the product family includes on and off automatic timers for efficient work scheduling. In addition, audible alarms help protect samples by alerting users to temperatures exceeding the defined range, and the icon-based graphical user interface has four preset options for easy navigation and use. The new Precision portfolio includes general purpose, shaking, circulating and coliform water baths. Each line has slightly different features to meet various application needs, but all are easy to maintain and clean with their coil-free interior. Other product features include low fluid protection for increased safety, and a gable cover that can be hinged open when using the bath.


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No statistical methods were used to predetermine sample size. Experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment. The recombineering technique21 was adapted to construct all targeting vectors for homologous recombination in ES cells. Retrieval vectors were obtained by combining 5′ miniarm (NotI/SpeI), 3′ miniarm (SpeI/BamHI) and the plasmid PL253 (NotI/BamHI). SW102 cells21 containing a BAC encompassing the carboxy-terminal part of the gene encoding the remodeller, were electroporated with the SpeI-linearized retrieval vector. This allowed the subcloning of genomic fragments of approximately 10 kilobases (kb) comprising the last exon of the gene encoding each remodeller. The next step was the insertion of a TAP-tag into the subcloned DNA, immediately 3′ to the coding sequence. The TAP-tag was (Flag) -TEV-HA for Chd1, Chd2, Chd4, Chd6, Chd8, Ep400, Brg1 and 6His-Flag-HA for Chd9. We first inserted the TAP-tag and an AscI site into the PL452 vector, to clone 5′ homology arms as SalI/AscI fragments into the PL452TAP-tag vector. 46C ES cells were electroporated with NotI-linearized targeting constructs and selected with G418. In all cases, G418-positive clones were screened by Southern blot. Details on the Southern genotyping strategy, as well as sequences of primers and plasmids used in this study are available on request. Correctly targeted ES cell clones were karyotyped, and the expression of each tagged remodeller was controlled by western blot analysis, using antibodies against Flag and haemagglutinin (HA) epitopes (see Extended Data Fig. 6). We also verified by immunofluorescence, using monoclonal antibodies anti-Flag (M2, Sigma F1804) and anti-HA (HA.11, Covance MMS-101P) epitopes, that each tagged remodeller was properly localized in the nucleus of ES cells. ES cell lines expressing a tagged remodeller were all indistinguishable in culture from their mother cell line (46C). Pluripotency of tagged ES cell lines was verified by detecting alkaline phosphatase activity on ES cell colonies 5 days after plating, using the Millipore alkaline detection kit, following manufacturer’s instructions. In addition, we verified by immunofluorescence using an antibody against Oct4 (also known as Pou5f1) (Abcam ab19857, lot 943333) that expression of this pluripotency-associated transcription factor was uniform in each tagged ES cell line. Mouse 46C ES cells have been described previously22. 46C ES cells and their tagged derivatives were cultured at 37 °C, 5% CO , on mitomycin C-inactivated mouse embryonic fibroblasts, in DMEM (Sigma) with 15% fetal bovine serum (Invitrogen), l-glutamine (Invitrogen), MEM non-essential amino acids (Invitrogen), penicillin/streptomycin (Invitrogen), β-mercaptoethanol (Sigma), and a saturating amount of leukaemia inhibitory factor (LIF), as described previously23. Mouse ES nucleosomal tags were acquired from a published MNase-seq data set7 to make the reference map shown in Fig. 2. Reference nucleosomes were called using MACS 2.0 before assigning the first MNase-resistant nucleosome upstream and downstream of TSSs as −1 and +1, respectively. Because long NFRs may actually contain MNase-sensitive nucleosome-like structures or histone-containing complexes, defining the first downstream MNase-resistant nucleosome as ‘+1’ is problematic, and so we refer to it as the ‘first stable nucleosome’. Regions between the associated −1 and +1 (or first stable) nucleosomes were defined as NFRs. We further defined narrow and wide NFR categories, which have the median width of 28 bp and 808 bp, respectively. We define HFRs as lacking histones as defined by ChIP-seq. The list of 14,623 genes used in Figs 1 and 2 was obtained by filtering all mm9 RefSeq genes24. We removed redundancies (that is, genes having the same start and end sites), unmappable genes, blacklisted genomic regions (those with artefact signal regardless of which NGS techniques were used), and genes shorter than 2 kb. The purpose of this last filtering step was to unambiguously distinguish the promoter region from the end of the genes in heat maps. Lists of genes defined as having H3K4me3 and bivalent promoters: we first defined, among the 14,623 RefSeq genes, those with a promoter that was positive for H3K4me3 (accession number: GSM590111). This was accomplished by operating with the seqMINER platform. Tag densities from this data set were collected in a −500/+1,000-bp window around the TSS, and subjected to three successive rounds of k-means clustering, to remove all genes with a promoter that was clustered with low H3K4me3. We next conducted on this series of H3K4me3-positive promoters three successive rounds of k-means clustering, using several published data sets for H3K27me3. The genes with a promoter positive for H3K27me3 in four distinct H3K27me3 data sets (accession numbers: GSM590115, GSM590116, GSM307619 and GSM392046/GSM392047) were considered as bivalent. We eventually obtained a list of 6,481 genes with H3K4me3-only promoters, and a list of 3,411 bivalent genes. A detailed version of this protocol is available on the protocol exchange website: http://dx.doi.org/10.1038/protex.2014.040. In brief, about 400 million ES cells were fixed either with formaldehyde, or with a combination of disuccinimidyl glutarate (DSG) and formaldehyde (Supplementary Table 1), then permeabilized with IGEPAL, and incubated with 2,800 units of micrococcal nuclease (MNase, New England Biolabs) in order to fragment the genome into mononucleosomes (Extended Data Fig. 1). This nucleosome preparation was next incubated with agarose beads coupled with an antibody anti-HA or anti-Flag. Anti-HA-agarose (ref. A2095) and anti-Flag-agarose (ref. A2220) beads were purchased from Sigma. After a series of washes, tagged remodeller–nucleosome complexes were eluted, either by TEV protease cleavage or by peptide competition (Supplementary Table 1). The eluted complexes were then subjected to a second immunopurification step, using beads coupled to the antibody specific of the second HA or Flag epitope. After elution, DNA was extracted from the highly purified mononucleosome fraction, and processed for high-throughput sequencing (see below). As a negative control, chromatin from untagged ES cells was subjected to the same protocol to define background signal. Two biological replicates were used for each tagged and control ES cell line, using independent cell cultures and chromatin preparations. After crosslink reversion, phenol–chloroform extraction and ethanol precipitation, the DNA from remodeller–nucleosome complexes was quantified using the picogreen method (Invitrogen) or by running 1/20 of the ChIP material on a high sensitivity DNA chip on a 2100 Bioanalyzer (Agilent). Approximately 5–10 ng of ChIP DNA was used for library preparation according to the Illumina ChIP-seq protocol (ChIP-seq sample preparation kit). Following end-repair and adaptor ligation, fragments were size-selected on an agarose gel in order to purify nucleosome-sized genomic DNA fragments between 140 and 180 bp. Purified fragments were next amplified (18 cycles) and verified on a 2100 Bioanalyzer before clustering and single-read sequencing on an Illumina Genome Analyzer (GA) or GA II, according to manufacturer’s instructions. Sequencing characteristics are shown in Supplementary Table 1. Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Ep400 and Brg1 MNase remodeller ChIP-seq short reads were mapped to mouse mm9 genome using Bowtie 0.12.7 with the followings settings: -a -m1–best–strata -v2 -p3. Data sets were next converted to BED format files, and data analysis was performed using the seqMINER platform25 (Fig. 1c). To examine the distribution of remodellers at individual genes, we used WigMaker3 (default settings) to convert BED files into wig files, which were uploaded onto the IGV genome browser (Extended Data Fig. 2). Nucleosome calls were made from MNase remodeller ChIP-seq tags using GeneTrack26 with the following parameters: sigma = 20, exclusion = 146. We then globally shifted tags to the median value of half distances of all nucleosome calls. GRO-seq tags10 sharing the same or opposite orientation with the TSS were assigned as ‘sense’ and ‘divergent’ tags, respectively. The orientation of each NFR was arranged so that sense transcription proceeds to the right. ES nucleosomal tags, globally shifted tags from MNase remodeller ChIP-seq (this current study), tags from DHS regions (Mouse ENCODE), GRO-seq oriented tags from transcriptionally engaged Pol II and CpG islands (UCSC, mm9 build) were then aligned to the midpoint of each NFR. Promoter regions were then sorted by NFR length and visualized by Java TreeView (Fig. 2a, b). CpG island information was retrieved from UCSC (mm9 build) and assigned to the closest TSS by using bedtools. We noticed that promoters with wide NFRs were mostly CpG island (CpGI)-rich, while those with narrow NFRs were globally CpGI-poor, in agreement with a previous report showing that CpGIs induce nucleosome exclusion9 (Fig. 2b). Tags from reference nucleosomes7, remodeller-interacting nucleosomes (this study) and transcriptionally engaged Pol II (GRO-seq)10 were aligned to nucleosome −1 and +1 (or the first stable nucleosome) dyad positions. The direction of each dyad was assigned according to the orientation of its associated TSS, the orientation of which was arranged so that the transcription proceeds to the right. After normalization to the gene count in the two different NFR subclasses, tags were plotted from the NFR midpoint to 500 bp distal to the reference nucleosome. An x axis gap in the NFR was introduced to normalize variations in NFR length inside each class. We used DNaseI-Seq data from the mouse ENCODE consortium (GSM1004653) for the identification of DHS regions in the mouse ES cell genome. DHS regions were defined using MACS 2.0 (ref. 27) (default setting), which resulted in the identification of 139,454 DHS regions. Each of these DHS regions was represented as a 500-bp window (−250 bp/+250 bp) centred on the midpoint of the DHS peak. DHS regions overlapping with the blacklisted (high background signal) genomic areas (mm9) were removed, resulting in a final list of 138,582 DHS regions. Tags from each tested ChIP-seq data set were summed up for each DHS region before pair-wise Pearson correlation comparison. The R2 value from each pair-wise Pearson correlation was then visualized by heat map (Fig. 1a). Pearson correlation analysis at promoter-like DHS regions. Operating with the seqMINER platform, we retrieved, from the 138,582 DHS regions list, those positive for H3K4me3, TBP and Pol II S5ph. We obtained 16,300 promoter-like DHS regions befitting the criteria. Pair-wise Pearson correlation was performed and plotted (Fig. 1b) as described for Fig. 1a. We used the pHYPER shRNA vector for remodeller depletion in ES cells, as previously described28. shRNA design was performed using DSIR software (http://biodev.extra.cea.fr/DSIR/DSIR.html). Below are the shRNAs selected for each remodeller. The sense strand sequence is given; the rest of the shRNA sequence is as described previously28. Chd1 shRNA 1: 5′-GCAAAGACGGCGACTAGAAGA-3′; Chd1 shRNA 2: 5′-GACAGTGCTTAATCAAGATCG-3′; Chd4 shRNA 1: 5′-GGACGACGATTTAGATGTAGA-3′; Chd4 shRNA 2: 5′-GCTGACGTCTTCAAGAATATG-3′; Chd6 shRNA 1: 5′-GTACTATCGTGCTATCCTAGA-3′; Chd6 shRNA 2: 5′-CAGTCAGAACCCACAATAACT-3′; Chd8 shRNA 1: 5′-GCAGTTACACTGACGTCTACA-3′; Chd8 shRNA 2: 5′-GACTTTCTGTACCGCTCAAGA-3′; Chd9 shRNA 1: 5′-TATACCAATTGAACAAGAGCC-3′; Chd9 shRNA 2: 5′-AGTTAAAGTCTACAGATTAGT-3′; Ep400 shRNA 1: 5′-GGTAAAGAGTCCAGATTAAAG-3′; Ep400 shRNA 2: 5′-GGTCCACACTCAACAACGAGC-3′; Smarca4 shRNA 1: 5′-ACTTCTTGATAGAATTCTACC-3′; Smarca4 shRNA 2: 5′-CCTTCGAACAGTGGTTCAATG-3′. Each shRNA was transfected in its corresponding tagged ES cell line, to follow remodeller depletion by western blotting using monoclonal antibodies anti-Flag (M2, Sigma F1804), or anti-HA (H7, Sigma H3663) epitopes (Extended Data Fig. 6), in comparison with the signal obtained with a control antibody anti-Gapdh (Abcam ab9485). The pHYPER shRNA vectors were transfected in ES cell by electroporation, using an Amaxa nucleofector (Lonza). Twenty-four hours after transfection, puromycin (2 μg ml−1) selection was applied for an additional 48 h period, before cell collection and RNA preparation, except for Chd4, for which cells were collected after 30 h of selection. Total RNA was extracted using an RNeasy kit (Qiagen). Total RNA yield was determined using a NanoDrop ND-100 (Labtech). Total RNA profiles were recorded using a Bioanalyzer 2100 (Agilent). For each remodeller, RNA was prepared from three independent transfection experiments, and processed for transcriptome analysis. 46C ES cells were amplified on feeder cells except for the last passage, at which point cells were plated onto 60-mm dishes coated with gelatine, and grown to 70% confluence in D15 medium with LIF. Total RNA was extracted using an RNeasy Kit (Qiagen). The RNA quality was verified on a 2100 Bioanalyzer. Library preparation was performed using the Illumina mRNaseq sample preparation kit according to manufacturer’s instructions. Briefly, the total RNA was depleted of ribosomal RNA using the Sera-mag Magnetic Oligo (dT) Beads (Illumina) and after mRNA fragmentation, reverse transcription and second strand cDNA synthesis the Illumina specific adaptors were ligated. The ligation product was then purified and enriched with 15 cycles of PCR to create the final library for single-read sequencing of 75 bp carried out on an Illumina GAIIx. To keep only sequences of good quality, we retained the first 40 bp of each read and discarded all sequences with more than 10% of bases having a quality score below 20, using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Mapping of these sequences onto the mm9 assembly of mouse genome and RPKM computation were then performed using ERANGE v3.1.0 (ref. 29) and bowtie v0.12.0 (ref. 30). In brief, a splice file was created with UCSC known genes and maxBorder = 36. We created an expanded genome containing genomic and splice-spanning sequences using bowtie-build and bowtie was used to map the reads onto this expanded genome. Then the ERANGE runStandardAnalysis.sh script was used to compute RPKM values following steps previously described29, using a consolidation radius of 20 kb. Random-primed reverse transcription was performed at 52 °C in 20 μl using Maxima First strand cDNA synthesis kit (Thermo Scientific) with 1 μg of total RNA isolated from ES cells (Qiagen), quantified with NanoDrop instrument (Thermo Scientific). Reverse transcription products were diluted 40-fold before use. Composition of quantitative PCR assay included 2.5 μl of the diluted RT reaction, 0.2–0.5 mM forward and reverse primers, and 1× Maxima SYBR Green qPCR Master Mix (Thermo Scientific). Reactions were performed in a 10 μl total volume. Amplification was performed as follows: 2 min at 95 °C, 40 cycles at 95 °C for 15 s and 60 °C for 60 s in the ABI/Prism 7900HT real-time PCR machine (Applied Biosystems). The real-time fluorescent data from qPCR were analysed with the Sequence Detection System 2.3 (Applied Biosystems). Each qPCR reaction was performed using the set of primer pairs listed in Supplementary Table 2, validated for their specificity and efficiency of amplification. All reactions were performed in triplicates, using RNA prepared from three independent cell transfection experiments. Control reactions without enzyme were verified to be negative. Relative expression was calculated after normalization with three reference genes (Actb, Nmt1 and Ddb1), validated for this study. cRNA was synthesized, amplified and purified using the Illumina TotalPrep RNA Amplification Kit (Life Technologies) following Manufacturer’s instructions. In brief, 200 ng of RNA were used to prepare double-stranded cDNA using a T7 oligonucleotide (dT) primer. Second-strand synthesis was followed by in vitro transcription in the presence of biotinylated nucleotides. cRNA samples were hybridized to the Illumina BeadChips Mouse WG-6v2.0 arrays. These BeadChips contain 45,281 unique 50-mer oligonucleotides in total, with hybridization to each probe assessed at 30 different beads on average. A total of 26,822 probes (59%) are targeted at RefSeq transcripts, and the remaining 18,459 (41%) are for other transcripts. BeadChips were scanned on the Illumina iScan scanner using Illumina BeadScan image data acquisition software (version 2.3). Data were then normalized using the ‘normalize quantiles’ function in the GenomeStudio Software (version 1.9.0). Following analyses were done using Genespring software (version 13.0-GX). For Brg1, we used a previously published transcriptome data set, in which loss of Brg1 function was obtained by genetic ablation18. All array analyses were undertaken using the Limma package from the R/Bioconductor software (R-Development-Core-Team, 2007). Microarray spot intensities were normalized using the RMA method as implemented in the R affy package. Normalized measures served to compute the log ratios for each gene between the wild-type strain and the Brg1 knockout mutant. Then, to identify genes with a log ratio significantly different between the mutant and wild- type strain, P values were calculated for each gene using a moderated t-test. The moderated t-test applied here was based on an empirical Bayes analysis and was equivalent to shrinkage (or expansion) of the estimated sample variances towards a pooled estimate, resulting in a more stable inference. Finally, adjusted P values were calculated using the false discovery rate (FDR)-controlling procedure of Benjamini and Hochberg. We identified deregulated genes using the thresholds of 0.05 for the P value, and 1.5 for the fold change (FC 1.5). This FC 1.5 threshold was chosen based on a previous study on Brg1 (ref. 18), and also because it was compatible with the analysis of the remodellers more modestly involved in transcriptional control in ES cells such as Chd1, Chd6 and Chd8. Note that seemingly modest fold changes might arise from many sources including a response lag, residual remodelling activity, and relatively high experimental background. Using a FC 2 threshold, we could, however, confirm that Ep400, Chd4 and Brg1 are important transcriptional regulators in ES cells, with 535, 293 and 570 genes deregulated, respectively. This level of deregulation is indicative of a context-specific function of remodellers in transcriptional activation or repression, which is distinct from the function of general transcription factors, whose depletion is expected to affect most genes. Statistical analysis of the differences in transcriptional activation and repression by remodellers was performed using a two-sample test for equality of proportions with continuity correction. For the generation of GC-content-based lists of promoters, we used the list of promoters defined in figure 3 of ref. 15, which we crossed with the 14,623 promoter list, to obtain a list of 6,317 promoters rank ordered according to GC content. In Fig. 3b, we compared the percentages of genes either down- or upregulated by loss of function of each remodeller in the following two groups: (1) NFR length classes: genes from the narrow and wide NFR classes shown in Fig. 2a were each further divided into two subclasses, which resulted in the following four categories: narrow NFR subclass 1 (NFR < 15 bp), narrow NFR subclass 2 (15–115 bp NFR), wide NFR subclass 1 (116–504 bp) and wide NFR subclass 2 (505–1,500 bp). Genes in these groups were further subdivided into H3K4me3 and bivalent subgroups. (2) GC content classes: genes were divided into four quartiles based on GC content at promoters and further subdivided into H3K4me3 and bivalent subclasses. The number of genes analysed in Fig. 3b is indicated in brackets for the following subgroups. H3K4me3 genes: narrow NFR subclass 1 (739), subclass 2 (1,829), wide NFR subclass 1 (2,613), subclass 2 (1,253), GC content quartile 1 (low GC content) (450), quartile 2 (719), quartile 3 (644), quartile 4 (high GC content) (430). Bivalent genes: narrow NFR subclass 1 (271), subclass 2 (866), wide NFR subclass 1 (2,266), subclass 2 (1,184), GC content quartile 1 (220), quartile 2 (485), quartile 3 (750) and quartile 4 (1149). FAIRE was performed as described31 with modifications. 46C ES cells were amplified as described above for RNA preparation. Formaldehyde was added directly to the growth media (final concentration 1%), and cells were fixed for 5 min at room temperature. After quenching with glycine (125 mM) and several washes, cells were collected, resuspended in 500 μl of cold lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) and disrupted using glass beads for five 1-min sessions with 2-min incubations on ice between disruption sessions. Samples were then sonicated for 16 sessions of 1 min (30 s on/30 s off) using a bioruptor (Diagenode) at max intensity, at 4 °C. After centrifugation, the supernatant was extracted twice with phenol–chloroform. The aqueous fractions were collected and pooled, and a final phenol–chloroform extraction was performed before DNA precipitation. FAIRE experiments were realized in triplicate, using independent ES cell cultures. Before sequencing, FAIRE DNA was analysed and quantified by running 1/25 of the FAIRE material on a high sensitivity DNA chip on a 2100 Bioanalyzer (Agilent, USA). Approximately 20 ng of FAIRE DNA was used for library preparation according to manufacturer’s instructions using the ChIP-seq sample preparation kit (Illumina). Single-read sequencing (36 bp) was performed on a Genome Analyzer II (Illumina). ES cells were grown and transfected with shRNA vectors as described for RNA analysis. Biological replicates were obtained by performing two independent transfection experiments for each shRNA vector. ATAC-seq libraries were constructed by adapting a published protocol20. In brief, 50,000 cells were collected, washed with cold PBS and resuspended in 50 μl of ES buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl ). Permeabilized cells were resuspended in 50 μl transposase reaction (1× tagmentation buffer, 1.0–1.5 μl Tn5 transposase enzyme (Illumina)) and incubated for 30 min at 37 °C. Subsequent steps of the protocol were performed as previously described20. Libraries were purified using a Qiagen MinElute kit and Ampure XP magnetic beads (1:1.6 ratio) to remove remaining adapters. Libraries were controlled using a 2100 Bioanalyzer, and an aliquot of each library was sequenced at low depth onto a MiSeq platform to control duplicate level and estimate DNA concentration. Each library was then paired-end sequenced (2 × 100 bp) on a HiSeq instrument (Illumina). As ATAC-seq libraries are composed in large part of short genomic DNA fragments, reads were cropped to 50 bp using trimmomatic-0.32 to optimize paired-end alignment. Reads were aligned to the mouse genome (mm9) using Bowtie with the parameters -m1-best-strata -X2000, with two mismatches permitted in the seed (default value). The -X2000 option allows the fragments <2 kb to align and -m1 parameter keeps only unique aligning reads. Duplicated reads were removed with picard-tools-1.85. To perform differential analysis, libraries were adjusted to 33 million aligned reads using samtools-1.2 and by making a random permutation of initial input libraries (shuf linux command line). Adjusted BAM data sets were next converted to BED. We used the seqMINER platform with the lists of 6,481 H3K4me3-only and 3,411 bivalent genes described above, to collect tag densities from ATAC-seq data sets, in a window of −2 kb/+2 kb around the TSS. Output tag density files were analysed using R software to establish average ATAC-seq signal profiles shown in Extended Data Fig. 8. ES cells were grown and transfected with shRNA vectors as described above. Biological replicates were obtained by performing two independent transfection experiments for each shRNA vector. For each experiment, 1 million cells were fixed 10 min in ES cell culture medium containing 1% formaldehyde, quenched with glycine (125 mM), washed with PBS buffer, collected in 175 μl of solution I (15 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl and 0.1 mM EGTA), and stored on ice. Cells were permeabilized by adding 175 μl of solution II (solution I with 0.8% Igepal CA-630 (Sigma)) and incubating for 15 min on ice. We next added 700 μl of MNase digestion buffer (50 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 15 mM KCl, 60 mM NaCl, 4 mM MgCl and 2 mM CaCl2), 4 U of MNase, and incubated for 10 min at 37 °C. MNase digestion was stopped by adding 10 mM EDTA (final concentration), and storing on ice. Cells were then disrupted by 15 passages through a 25 G needle, followed by a 10 min centrifugation at 18,000g. The supernatant was collected and incubated for 1 h at 65 °C with 15 μg of RNase A. We next added 10 μg of proteinase K, adjusted each sample to 0.1% SDS (final concentration) and incubated for 2 h at 55 °C. NaCl concentration was then adjusted to 200 mM and the samples were incubated overnight at 65 °C for crosslink reversal. DNA was purified from each sample by phenol–chloroform extraction followed by ethanol precipitation. Purified DNA (20 ng) was used for library preparation according to manufacturer’s instructions, using Ultralow ovation library system (Nugen). Following end-repair and adaptor ligation, fragments were size-selected onto an agarose gel in order to purify genomic DNA fragments between ~60 and 220 bp. Libraries were verified using a 2100 Bioanalyzer before clustering and paired-read sequencing. Sequencing of each sample was performed in a single lane of a HiSeq instrument (Illumina). The midpoint of each paired-end sequencing read was used to represent dyad location of each nucleosomal tag. We assumed that remodeller depletion has no bulk effect on nucleosome occupancy, hence the total reads of control and remodeller-depleted cells were adjusted to be the same. The adjusted tags were aligned to −1 nucleosome dyads (determined by the first MNase-defined peak upstream of annotated RefSeq TSS), or the first stable (MNase-defined) nucleosome dyad position downstream of the TSS for different NFR categories. These tags were further normalized to the amount of genes involved in each NFR class. The normalized tags were then binned (5 bp) and smoothed (10-bin moving average) before plotting (Fig. 3c). Distances (bp) are indicated relative to these reference points. An x axis gap in the NFR was introduced to normalize variations in NFR length inside each class. ES cells were grown and transfected with shRNA vectors as described above. Biological replicates were obtained by performing two independent transfection experiments for each shRNA vector. Following a 10 min fixation with 1% formaldehyde in ES cell culture medium, chromatin was prepared from 5–10 million cells and sonicated as described32. ChIP-exo experiments were carried out essentially as described33. This included an immunoprecipitation step using antibodies against Pol II (sc-899, Santa Cruz Biotechnology) attached to magnetic beads, followed by DNA polishing, A-tailing, Illumina adaptor ligation (ExA2), and lambda and recJ exonuclease digestion on the beads. After elution, a primer was annealed to EXA2 and extended with phi29 DNA polymerase, then A-tailed. A second Illumina adaptor was then ligated, and the products PCR-amplified and gel-purified. Sequencing was performed using NextSeq500. Uniquely aligned sequence tags were mapped to the mouse genome (mm9) using BWA-MEM (version 0.7.9a-r786)34. The uniquely aligned sequence tags were used for the downstream analysis. The 5′ end of mapped tags, representing exonuclease stop sites, were consolidated into peak calls (sigma = 5, exclusion = 20) using GeneTrack26, and peak pairs were matched when found on opposite strands and 0–100 bp apart in the 3′ direction. Tags were globally shifted to the median value of half distance between all peak pairs. These global shifted tags were then aligned relative to the annotated RefSeq TSSs for H3K4me3-only and bivalent promoters separately before further carved out remodeller-affected genes. We assumed that having remodeller deletion bore no bulk change on Pol II occupancy, and hence total tags among wild type and all remodeller mutants were normalized to be the same. To make direct comparison between different gene groups, we further normalized tags to the amount of genes within the group. These normalized tags were then smoothed (5 bp binned before 10-bin moving average) before plotting (Extended Data Fig. 9a). To examine Pol II occupancy change in remodeller mutants among different promoter groups, we first calculated total Pol II occupancy by summing up tags from transcript start to end sites (annotated RefSeq TSS and TES, respectively24) for the tested genes. Change in Pol II occupancy was calculated by dividing the total Pol II occupancy of mutant by that of wild type before log transformation and bargraph plotting (Extended Data Fig. 9b). Genes were rank-ordered according to reads per kb of transcript per million mapped reads (rpkm) and divided in four quartiles (highest: Q4, second: Q3, third: Q2 and lowest: Q1). Operating with the k-means clustering function of seqMINER, genes in each quartile were further subdivided in H3K4me3-only and bivalent genes, as described above. Using these lists of genes, tag densities from remodeller ChIP-seq data sets were collected in a window of −2 kb/+2 kb around the TSS, except for Chd2, for which densities were collected from the TSS until +4 kb. Output tag density files were first analysed using R software to establish average binding profiles. Statistical comparisons were performed between remodeller distributions at H3K4me3 promoters, to assess a significant increasing trend among distributions. Differences between successive pairs of quartiles (Q4 − Q3, Q3 − Q2 and Q2 − Q1) were compared against a null distribution using a one side t-test. The respective P values are reported for each remodeller: Chd1, Q4 − Q3 P = 1.371138 × 10−27; Q3 − Q2 P = 1.728126 × 10−16; Q2 − Q1 P = 7.985217 × 10−23. Chd2, Q4 − Q3 P = 7.543473 × 10−33; Q3 − Q2 P = 1.115223 × 10−25; Q2 − Q1 P = 3.283427 × 10−38. Chd4, Q4 − Q3 P = 0.2094255; Q3 − Q2 P = 0.1081455; Q2 − Q1 P = 0.07202865. Chd6, Q4 − Q3 P = 0.4168748; Q3 − Q2 P = 0.1534144; Q2 − Q1 P = 0.01138035. Chd8, Q4−Q3 P = 4.031959 × 10−15; Q3 − Q2 P = 1.231527 × 10−6; Q2 − Q1 P = 1.34455 × 10−9. Chd9, Q4 − Q3 P = 9.484578 × 10−44; Q3 − Q2 P = 1.059783 × 10−14; Q2 − Q1 P = 4.646352 × 10−28. Ep400, Q4 − Q3 P = 3.046796 × 10−20; Q3 − Q2 P = 1.215304 × 10−14; Q2 − Q1 P = 6.462667 × 10−11. Brg1, Q4 − Q3 P = 3.512021 × 10−24; Q3 − Q2 P = 2.515217 × 10−7; Q2 − Q1 P = 0.977422. We concluded from this analysis that Chd1, Chd2, Chd9 and Ep400 binding at promoters is tightly linked to gene expression level. By contrast, Brg1, Chd4 and Chd6 deposition showed little correlation with gene expression level (statistical test failed for at least one comparison for these remodellers). While statistical analysis of Chd8 distributions concluded to significant differences between quartiles, inspection of distributions in Extended Data Fig. 3 showed that Chd8 binding profile was intermediate between these two categories.


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Caesium carbonate (99.995%, trace metal basis), 2-thiophenecarboxylic acid (99%), benzoic acid-(phenyl-13C ) (99 at% 13C), and tetrabutylammonium bromide (TBABr, 99%) were purchased from Sigma Aldrich; caesium carbonate (≥99%, for large-scale reactions) and furan-2,5-dicarboxylic acid (99.6%) were purchased from Chem Impex International; dimethyl furan-2,5-dicarboxylate (99%) was purchased from Astatech; benzoic-d acid-(phenyl-d ) (98%) was purchased from Cambridge Isotope Laboratories; 2-furoic acid (98%), benzoic acid (99%) and anhydrous methyl alcohol (99.8%) were purchased from Acros Organics; sodium L-(+)-tartrate dihydrate (99.7%), benzene (HPLC grade) was purchased from Alfa Aesar; carbon dioxide (99.99%) was purchased from Praxair. The methanol was further dried over 3 Å molecular sieves before using. Reagent-grade benzene was dried by passing through an activated alumina column on an Innovative Technology PureSolv solvent purification system. N,N-dimethylformamide (DMF, 99.8%) was purchased from Fisher Scientific and dried by passing through an activated molecular sieve column. All other chemicals were used as received without further purification. Experiments under flowing CO were performed in a Thermo Scientific Lindberg/Blue M tube furnace. Experiments under pressurized CO or N were performed in a 300-ml high-temperature, high-pressure Parr reactor (model 4561-HT-FG-SS-115-VS-2000-4848) equipped with a glass liner. 1H-NMR, 2H-NMR and 13C-NMR spectra were recorded at 23 °C on a Varian Unity Inova 600 MHz spectrometer, a Varian Unity Inova 500 MHz spectrometer, a Varian Direct Drive 400 MHz spectrometer, a Varian Mercury 400 MHz spectrometer, or a Varian Unity Inova 300 MHz spectrometer. 1H chemical shifts (δ) are reported in parts per million downfield from tetramethylsilane and referenced to residual protium in the NMR solvent (D O, δ = 4.79, CDCl , δ = 7.26). 2H chemical shifts (δ) are reported in parts per million downfield from tetramethylsilane and referenced to deuterium in the NMR solvent (D O, δ = 4.71). 13C chemical shifts (δ) are reported in parts per million downfield from tetramethylsilane and referenced to carbon resonances in the NMR solvent (CDCl δ = 77.16, centre line), to added methanol (δ = 49.00), or to added tetramethylsilane (δ = 0.00). High-resolution mass spectra were recorded on a Waters Aquity UPLC and Thermo Exactive Orbitrap mass spectrometer by direct injection electrospray ionization–mass spectrometry (ESI-MS). The carboxylic acid (2-furoic acid, thiophene-2-carboxylic acid or benzoic acid) and 1.05 equivalent of Cs CO were dissolved in the minimum amount of deionized water and evaporated to dryness by heating at 150 °C for at least 2 h. The solid mixture was cooled and used directly for C–H carboxylation reactions. We note that residual moisture in the reactant mixture reduces the yield of the carboxylation reaction. The appropriate amount of reactant mixture was weighed out into a quartz boat and placed in the tube furnace. The furnace was heated to the desired temperature under CO flowing at 40 ml min−1 for a given period of time. The sample was cooled to ambient temperature, dissolved in D O and filtered through a 0.2 μm polytetrafluoroethylene (PTFE) syringe filter to prepare samples for NMR analysis. The product yields were calculated from integration of the 1H NMR peaks using sodium L-(+)-tartrate dihydrate as an internal standard. Representative spectra and data are shown in Extended Data Figs 1 and 2 and Extended Data Table 1a. The reactant mixture was charged into the Parr reactor equipped with a glass liner. The reactor was sealed and then evacuated and backfilled with CO three times. It was then filled to a final CO pressure at ambient temperature corresponding to a pressure of 8 bar at the desired reaction temperature. The reactor was heated to the desired temperature, maintained at that temperature for a given period of time, and then cooled to ambient temperature and depressurized. The solid product mixture was dissolved in D O and analysed using NMR as described above. Representative spectra and data are shown in Extended Data Figs 3 and 4 and Extended Data Table 1a. To a 1-litre round-bottomed flask we added the 2-furoic acid (100 mmol, 11.21 g, 1.0 equiv.) and Cs CO (125 mmol, 40.73 g, 1.25 equiv.) followed by 100 ml of deionized H O. The addition of water results in an acid–base reaction that liberates CO . (Caution: this reaction is exothermic and effervescent.) Once the reaction was complete, the water was removed in vacuum on a rotary evaporator (rotovap) at 75 °C and at 100 mTorr and 230 °C. The resulting off-white solid was scraped and ground into a fine white powder. In a fume hood, a reactor was assembled consisting of a rotovap with P O in the collection flask connected to a Schlenk line and a eutectic salt bath (48.7 mol% NaNO , 51.3 mol% KNO ). The eutectic salt bath was heated to 260 °C, and the 1-litre round-bottomed flask containing the caesium furan-2-carboxylate and Cs CO was attached to the rotovap. The joint was taped with black electrical tape and fitted with a green Keck clip, and the entire apparatus was the backfilled with CO gas. A short piece of tubing was connected in such a way as to deliver a slow stream of cooling air to the taped joint to prevent melting. The reaction was then dipped into the eutectic salt bath and rotated slowly for 48 h with a gentle flow of CO through the bubbler of the Schlenk line. Over the course of the reaction, the solid initially melts, then turns blackish-brown and solidifies. Once complete, the reaction was slowly cooled to room temperature and detached from the rotovap. Disodium tartrate dihydrate (10 mmol, 2.31 g, 0.1 equiv.) was added followed by 200 ml of deionized water. An aliquot of the resulting solution was evaporated in vacuum then dissolved in D O. A 1H NMR was obtained with the following distribution of products: caesium furan-2,5-dicarboxylate (71%), caesium malonate (2%), and caesium acetate (11%) (NMR yields). A repeat of the experiment gave the following distribution: caesium furan-2,5-dicarboxylate (69%), caesium malonate (7%), and caesium acetate (8%). The product was isolated from the reaction mixture using the following procedure. First, the reaction was filtered through a pad of celite to remove insoluble material. The resulting solution was then acidified below a pH of 2 with sulfuric acid (15 ml concentrated acid). (Caution: this reaction is exothermic and effervescent.) The product precipitated from the solution and was collected on a Büchner funnel. The product was then dissolved in 1 litre of methanol and decolorized with activated carbon. The activated carbon was removed by filtering the solution through a pad of celite. The resulting solution was concentrated in vacuum and then triturated with 500 ml of ethyl acetate. The product was collected on a Büchner funnel, washed with ethyl acetate, and then dried in vacuum to afford a white crystalline powder (10.35 g, 66%). 2-furoic acid (56 mg, 0.50 mmol, 1.0 equiv.), potassium isobutyrate (63 mg, 0.50 mmol, 1.0 equiv.), and K CO (73 mg, 0.53 mmol, 1.05 equiv.) were dissolved in the minimum of water in a quartz boat, and evaporated to dryness by heating at 150 °C for 2 h under a stream of N . The sample was heated to 320 °C under CO flowing at 40 ml min−1 in the tube furnace for 8 h. The solid product mixture was dissolved in D O and analysed using 1H NMR as described above. The yield of potassium FDCA2– was 62%, and the conversion of furan-2-carboxylate was 76%. A representative spectrum is shown in Extended Data Fig. 5a. A carboxylation reaction was also performed with 2-furoic acid (56 mg, 0.50 mmol, 1.0 equiv.), potassium acetate (49 mg, 0.50 mmol, 1.0 equiv.), and K CO (73 mg, 0.53 mmol, 1.05 equiv.). The sample was heated to 300 °C under CO flowing at 40 ml min−1 in the tube furnace for 8 h. The yield of potassium FDCA2– was 57%, and the conversion of furan-2-carboxylate was 96%. A two-necked 25-ml round-bottomed flask was equipped with a reflux condenser, gas adaptor, PTFE-coated stir-bar, and a septum. The flask was charged with 2-furoic acid (115 mg, 1.03 mmol, 1.03 equiv.), Cs CO (555 mg, 1.70 mmol, 1.70 equiv.), and water (2 ml). Once the resulting acid–base reaction subsided, the reaction vessel was heated to 150 °C in an oil bath under a stream of N . After 30 min the solution had dried out, and the reactor was then placed under vacuum and heated to 175 °C. At the same time, a heat gun was used to dry the rest of the apparatus. The reactor was then cooled to 125 °C and back-filled three times with CO . Using a syringe, dry DMF (2 ml) was added to the reaction. The reaction was then stirred for 12 h at 125 °C under a CO atmosphere (1 bar). After 12 h, the reaction was concentrated under vacuum. NMR analysis of the residue in D O indicated no conversion of the starting material. Into a 20 ml vial we added a mixture of caesium carbonate (489 mg, 1.5 mmol, 1.0 equiv.) and caesium isobutyrate (220 mg, 1.0 mmol, 0.67 equiv.). This vial was placed into the Parr reactor, which was sealed and then evacuated and backfilled with CO three times. Anhydrous benzene was injected into the reactor in an amount ranging from 10 ml to 35 ml depending on the desired final partial pressure of benzene. The reactor was then pressurized with CO and heated to the desired temperature. The partial pressure of CO at the final temperature was calculated assuming ideal behaviour. The partial pressure of benzene was calculated by subtracting the CO pressure from the measured reactor pressure. After the indicated period of time (Extended Data Table 1b), the reactor was cooled to ambient temperature and depressurized. The crude product was dissolved in D O and analysed using NMR as described above. In addition to benzene carboxylation products, isobutyrate decomposition products were observed, which included formate, acetate, acetate carboxylation products (malonate and methane tricarboxylate), and insoluble char. Control experiments were performed in the absence of either benzene or gaseous CO . In the absence of benzene, only a trace amount of formate was observed and the main product was insoluble char from isobutryrate decomposition. In the absence of gaseous CO , a small amount of benzoate was observed, which is attributed to the formation of CO in situ from the decomposition of HCO – that is formed by deprotonation of benzene with CO 2–. Representative spectra and data are shown in Extended Data Fig. 5b and Extended Data Table 1b. For comparison, an experiment was performed in the absence of caesium isobutyrate. A 20 ml vial with caesium carbonate (326 mg, 1.0 mmol) was placed into the Parr reactor, which was sealed and then evacuated and backfilled with CO three times. 35 ml of anhydrous benzene was injected into the reactor. The reactor was pressurized with 15 bar of CO and heated to 340 °C or 380 °C for 3 h and 8 h respectively. The reactor was then cooled to ambient temperature and depressurized. The crude product was dissolved in D O and analysed using NMR as described above. No reaction was observed in this case. The Parr reactor was equipped with an oven-dried glass liner and charged with caesium furan-2-carboxylate (244 mg, 1.0 mmol). The reactor was sealed and then evacuated and backfilled with CO three times. It was pressurized to 5 bar CO and then heated to 200 °C, at which point the CO pressure was 8 bar. After 2 h, the reactor was cooled to room temperature, vented, and disassembled. The residue was dissolved in D O and analysed using NMR. No FDCA2– was formed in the reaction. The general procedure outlined previously (see sections ‘Preparation of reactant mixtures consisting of caesium carboxylate + 0.55 equivalents Cs CO ,’ and ‘C–H carboxylation under pressurised CO ’) was followed using 2-furoic acid (10 mmol, 1.12 g, 1.00 equiv.) and Cs CO (10.5 mmol, 3.42 g, 1.05 equiv.). Once the reaction had completed and cooled to room temperature, the resulting solid was treated with 7 ml of 3 N HCl. The FDCA immediately precipitated from the reaction mixture as an off-white solid. The suspension was filtered through a glass frit (medium porosity) and washed with a minimum of deionized water (3 × 0.5 ml). The filtrate was then transferred to a 100-ml round-bottomed flask and the filter cake was transferred, washing with methanol, to a separate, tared, 100-ml round-bottomed flask. The flask containing the filter cake was evaporated to dryness to afford a yellow solid. The solid was analysed using 1H NMR in acetone-d . NMR analysis indicated a crude isolated yield of 81% for FDCA, along with 8% residual unreacted 2-furoic acid. The flask containing the filtrate was evaporated to dryness to afford 3.74 g yellow solid. The solid was analysed using 1H NMR in D O with an internal standard to quantify organic contaminants. 2-furoic acid and FDCA were present in an amount corresponding to <0.4% of the mass of the solid. Based on this analysis, the caesium was recovered in >99% yield as the CsCl salt. The general procedure outlined previously (see section ‘100-mmol-scale synthesis of furan-2,5-dicarboxylic acid at 1 atm of CO ’) was followed using the following quantities of 2-furoic acid (100 mmol, 11.23 g, 1.00 equiv.) and Cs CO (105 mmol, 34.36 g, 1.05 equiv.). Once the reaction had completed and cooled to room temperature, the resulting solid was treated with 110 ml of 2 N HCl. The FDCA immediately precipitated from the reaction mixture as an off-white solid. This reaction was filtered through a glass frit (medium porosity) and washed with a minimum of deionized water (3 × 5 ml). The filtrate was then transferred to a tared 500-ml round-bottomed flask and the filter cake was transferred and washed with methanol into a separate, tared, 500-ml round-bottomed flask. The filter cake and filtrate were massed and analysed as described above, yielding the following results: 69% crude isolated yield for FDCA; >98% recovery of caesium as CsCl. 2-Furoic acid (112 mg, 1.0 mmol), acetic acid-d (58 μl, 1.0 mmol) and Cs CO (682 mg, 2.1 mmol) were dissolved in the minimum amount of deionized water and evaporated to form a dry powder that consisted of 1 mmol caesium furan-2-carboxylate, 1 mmol caesium acetate-d and 1.1 mmol of Cs CO . This material was heated in the Parr reactor to 200 °C under 2 bar of N for 1 h. After cooling to room temperature, the product mixture was dissolved in D O and analysed by NMR. The integration of the furan-2-carboxylate peaks in the 1H NMR spectrum using an internal standard indicated that the H/D scrambling was ~60% complete with substantially more scrambling at the 5 position than at the 3 and 4 positions. The presence of D at all positions was evident in the 2H NMR spectrum, the peak splitting of the 13C NMR spectrum, and in the high-resolution mass spectrum (Fig. 2d and Extended Data Figs 6b, 7a and 7b). For comparison, an experiment was performed in the absence of Cs CO . 2-Furoic acid (112 mg, 1.0 mmol), acetic acid-d (58 μl, 1.0 mmol) and Cs CO (312 mg, 0.96 mmol) were dissolved in minimum amount of deionized water and evaporated to form an oil that consisted of caesium furan-2-carboxylate and caesium acetate-d . A 1H NMR spectrum of this mixture is shown in Extended Data Fig. 6a. This material was heated in the Parr reactor to 200 °C under 2 bar of N for 1 h. After cooling to room temperature, the material was dissolved in D O and analysed by NMR. Integration of the furan-2-carboxylate peaks in the 1H NMR spectrum using an internal standard indicated that the H/D scrambling was ~15% complete with substantial scrambling at the 5 position and almost no scrambling at the 3 and 4 positions. The presence of D at the 5 position was evident in the 2H NMR spectrum, the peak splitting of the 13C NMR spectrum, and in the high-resolution mass spectrum (Fig. 2d and Extended Data Fig. 8). The mass spectrometry sample was prepared by adding 6 N HCl dropwise to the NMR sample until the clear solution turned into a suspension. The water was removed under vacuum and the residue was suspended in 2.5 ml of methanol. The suspension was allowed to stand till the solid particles settled. An aliquot of the supernatant liquid was further diluted with methanol and analysed directly by mass spectrometry. Benzoic acid-(phenyl-13C ) (7.8 mg, 60.9 μmol), benzoic-d acid-(phenyl-d ) (7.8 mg, 61.3 μmol) and Cs CO (41.5 mg, 127.4 μmol) were dissolved in 1 ml of deionized water and evaporated to form a dry powder that consists of caesium benzoate-(phenyl-13C ), caesium benzoate-(phenyl-d ) and 0.55 equivalents of Cs CO . This material was heated in the Parr reactor to 320 °C under 2 bar of N for 1 h. After cooling to room temperature, the material was dissolved in D O and analysed by NMR. The 1H spectra of the reactant mixture and the product mixture are shown in Fig. 3. A control experiment was performed to test whether Cs CO is necessary for isotopic scrambling. Benzoic acid-(phenyl-13C ) (7.8 mg, 60.9 μmol), benzoic-d acid-(phenyl-d ) (7.8 mg, 61.3 μmol) and Cs CO (19.8 mg, 60.7 μmol) were dissolved in 1 ml H O and evaporated to form a dry powder that consists of the caesium benzoate salts. After heating to 320 °C under 2 bar N for 1 h, no H/D exchange was observed by 1H NMR (Extended Data Fig. 9). Caesium FDCA2– (420 mg, 1.0 mmol) was charged into the Parr reactor equipped with an oven-dried glass liner. The reactor was sealed and then evacuated and backfilled with CO three times. Anhydrous methanol (100 ml) was injected into the reactor. The reactor was then pressurized with either 28.5 bar or 15 bar CO and heated to 200 °C or 180 °C. After 30 min, the reactor was cooled to ambient temperature, vented, and disassembled. The reaction mixture was transferred to a 250-ml round-bottomed flask and the methanol was removed under vacuum on a rotary evaporator at 45 °C. The residue was washed twice with 5 mL CHCl to dissolve the DMFD. The combined CHCl washes were evaporated to afford DMFD as a white powder. The material was dissolved in CDCl and analysed by 1H NMR with TBABr as an internal standard. The remaining residue that was not dissolved in the CHCl washes was dissolved in CD OD and analysed by 1H NMR using TBABr as an internal standard. This material consists of FDCA2−, MMFD, and a small amount of additional DMFD. DMFD (184 mg, 1.0 mmol, 1 equiv.) and Cs CO (326 mg, 1.0 mmol, 1 equiv.) were charged into a Parr reactor equipped with an oven-dried glass liner. The reactor was sealed and then evacuated and backfilled with CO three times. Anhydrous methanol (100 ml) was injected into the reactor. The reactor was pressurized with 28.5 bar CO and heated to 200 °C The total pressure at 200 °C was 105 bar and the calculated CO pressure was 45 bar. After 30 min, the reactor was cooled down to ambient temperature then vented and disassembled. The reaction mixture was transferred to a 250-ml round-bottomed flask and the methanol was removed under vacuum on a rotary evaporator at 45 °C. The residue was processed and analysed by 1H NMR as described above for the esterification of FDCA2–. The Parr reactor was equipped with an oven-dried glass liner and charged with caesium furan-2-carboxylate (244 mg, 1.0 mmol, 1.0 equiv.) and Cs CO (179 mg, 0.55 mmol, 0.55 equiv.). The reactor was sealed and then evacuated and backfilled with CO three times. The reactor was pressurized to 5 bar CO and then heated to 200 °C, at which point the CO pressure was 8 bar. After 5 h, the reactor was cooled and vented then evacuated and backfilled with N . To remove the water by-product, 10 ml of dry methanol was injected into the reactor to dissolve the reaction mixture. The methanol was removed by heating the reactor to 150 °C under vacuum. Subsequently, N gas was flowed over the reaction mixture for 8 h by keeping the gas release valve of the reactor open. The reactor was cooled to ambient temperature and 90 ml of dry methanol was injected into it. The vessel was pressurized with 28.5 bar CO and heated to 200 °C. After 30 min, the reactor was cooled to ambient temperature, depressurized and opened. The reaction mixture was diluted with 65 ml deionized water and extracted with CHCl (2 × 65 ml). The combined organic layers were dried over Na SO and concentrated under vacuum to afford the DMFD as a white solid and the yield was determined from 1H NMR using TBABr as an internal standard. The aqueous extract was concentrated under reduced pressure to approximately a 2 ml solution and transferred to the same glass liner used in the first cycle. To the solution was added 2-furoic acid equivalent to the amount of DMFD produced in the first cycle. The liner was resealed inside the reactor and heated to 150 °C under an atmosphere of N . The reactor was subsequently evacuated and backfilled with N , kept under a stream of N at 150 °C for 8 h, and then cooled to ambient temperature. The reaction mixture was subjected to a second cycle of carboxylation followed by esterification following the same procedures described above. At the completion of the cycle, the solution was diluted with 65 ml deionized water and extracted with CHCl (2 × 65 ml). The combined organic layers were dried over Na SO and concentrated under vacuum to afford the DMFD (Extended Data Fig. 10a). Evaporation of the aqueous extract yielded the crude mixture of Cs FDCA, caesium 5-(methoxycarbonyl)furan-2-carboxylate and Cs CO . The amount of unreacted Cs FDCA and caesium 5-(methoxycarbonyl)furan-2-carboxylate were quantified by 1H NMR using sodium L-(+)-tartrate dihydrate as an internal standard (Extended Data Fig. 10b). The NMR peaks in the spectra for the carboxylation reactions were assigned to different products by comparison to spectra for pure caesium salts obtained independently from the pure carboxylic acids. The resonances for these compounds are provided below for reference.


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No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. CENP-LN was produced as a GST fusion construct from insect cells using the MultiBac expression system31. Specifically, a coding sequence expressing 3C cleavable GST-tagged CENP-L was sub-cloned into MCS2, and the coding sequence of CENP-N was sub-cloned into MCS1 of pFL. Bacmid was then produced from EMBacY cells31, and subsequently used to transfect Sf9 cells and produce baculovirus. Baculovirus was amplified through three rounds of amplification and used to infect Tnao38 cells32. Cells infected with the GST- CENP-L/CENP-N virus were cultured for 72 h before harvesting. Cells were washed and resuspended in lysis buffer (50 mM Na-HEPES, 300 mM NaCl, 10% glycerol, 4 mM 2-mercaptoethanol, 1 mM MgCl pH 7.5). Resuspended cells were lysed by sonication in the presence of Benzonase before clearance at 100,000g at 4 °C for 1 h. Cleared lysate was passed over GSH-Sepharose, before extensive washing with lysis buffer. GST-CENP-L/CENP-N complex was then eluted in lysis buffer + 20 mM reduced glutathione. Eluted protein was concentrated in a 30 kDa Amicon-Ultra-15 Centrifugal Filter (Millipore) in the presence of GST-tagged 3C protease. Concentrated protein was then loaded onto a Superdex 200 16/600 column equilibrated in 20 mM Na- HEPES pH 7.5, 300 mM NaCl, 2.5% glycerol. A 5 ml GSH-Sepharose FF column was connected in series after the Superdex 200 column to trap GST, un-cut GST-CENP-L/CENP-N and GST-tagged 3C protease. Peak fractions corresponding to CENP-L/CENP-N were collected and again concentrated in a 30 kDa MWCO concentrator to approximately 50–100 μM before being flash frozen in liquid N and stored at −80 °C. Synthetic, codon-optimized DNA (Geneart), encoding the human CENP-C1–544His, CENP-C189–544, or CENP-C545–943 was sub-cloned into pFL or pFG (containing an N-terminal 3C cleavable GST) vectors, respectively, by restriction cloning with the enzymes BamHI and SalI. A non-cleavable histidine tag comprising six histidines (His6-tag) was introduced C-terminally of CENP-C1–544His, a tobacco etch virus (TEV) cleavage site was introduced N-terminal of CENP-C545–943. Tnao38 cells expressing CENP-C1–544His, CENP-C189–544, or CENP-C545–943 were resuspended in lysis buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 2 mM β-mercaptoethanol) and lysed by sonication before centrifugation at 100,000g at 4 °C for 1 h. The cleared lysates were incubated with Ni-NTA Agarose beads (for CENP-C1–544His), GST-Trap affinity column (GE Healthcare, for CENP-C189–544) or Glutathione Sepharose 4 Fast Flow beads (for CENP-C545–943) at 4 °C for 2 h. After washing with 70 column volumes of lysis buffer, CENP-C1–544His was eluted with lysis buffer supplemented with 200 mM Imidazole, CENP-C189–544 was eluted in lysis buffer supplemented with 30 mM reduced glutathione, and CENP-C545–943 was cleaved off the beads in 16 h at 4 °C by addition of TEV protease. After elution, proteins were diluted in buffer A (20 mM HEPES pH 7.5, 5% glycerol, 1 mM TCEP, to achieve a final concentration of 300 mM NaCl), loaded onto a pre-equilibrated HiTrap Heparin HP column, and eluted with a linear gradient of buffer B (20 mM HEPES pH 7.5, 2 M NaCl, 5% glycerol, 1 mM TCEP) in a gradient from 300 to 1200 mM NaCl. Fractions containing CENP-C1–544His and CENP-C545–943 were loaded onto a Superdex 200 16/60 SEC column pre-equilibrated in SEC buffer (10 mM HEPES pH 7.5, 300 mM NaCl, 2.5% glycerol, 2 mM TCEP). For CENP-C189–544, the GST tag was cleaved using 3C protease and the protein concentrated in a 10 kDa MWCO concentrator. The protein was then further purified by SEC as described for the other two constructs. SEC fractions containing CENP-C1–544His, CENP-C189–544, or CENP-C545–943 were concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C. NDC80-GFP complexes were constructed with a C-terminal fusion of GFP to HEC1. The unlabelled NDC80 complex was constructed with an N-terminal fusion of a His6-tag to SPC25. Construct for insect cell expression exploited the MultiBac baculovirus expression system31. Bacmid was then produced from EMBacY cells, and subsequently used to transfect Sf9 cells and produce baculovirus. Baculovirus was amplified through three rounds of amplification and used to infect Tnao38 cells. Cells infected with virus expressing untagged NDC80 were cultured for 72 h before harvesting. Cells were washed and resuspended in lysis buffer (25 mM Na-HEPES, 300 mM NaCl, 10% glycerol, 1 mM TCEP, 1 mM MgCl pH 7.5 and 1 mM PMSF). Resuspended cells were lysed by sonication in the presence of Benzonase before clearance at 100,000g at 4 °C for 1 h. Cleared lysate was passed over Ni-Sepharose, before extensive washing with lysis buffer. The Ndc80 complex was then eluted in lysis buffer + 250 mM imidazole. Eluted protein was diluted to 50 mM NaCl using buffer A (25 mM Na-HEPES, 10% glycerol, 1 mM TCEP) and loaded on a ResQ anion-exchange column. The NDC80-GFP was eluted using a salt gradient over 30 column volumes to 500 mM NaCl using buffer B (25 mM Na-HEPES, 1,000 mM NaCl, 10% glycerol, 1 mM TCEP). The eluted protein was concentrated in a 30-kDa Amicon-Ultra-15 Centrifugal Filter (Millipore) and the concentrated protein was then loaded onto a Superdex 200 16/600 column equilibrated in 10 mM Na- HEPES pH 7.5, 150 mM NaCl, 2.5% glycerol, pH 7.5. Peak fractions containing the NDC80 complex were collected and again concentrated in a 30 kDa MWCO concentrator to approximately 10 μM before being flash frozen in liquid N and storage at −80 °C. Codon-optimized human CENP-I57–756 (57-C) was subcloned in a MultiBac pFL-derived vector31 with an N-terminal TEV cleavable His6-tag, under the control of the polh promoter. A complementary DNA (cDNA) segment encoding human CENP-M isoform 1 was subcloned in the second MCS of the same vector, under the control of the p10 promoter. Simultaneously, a second pFL-based vector was created with untagged CENP-H and CENP-K under the control of the polh and p10 promoters, respectively. The CENP-I/M vector was then linearized with BstZ171, and the expression region of the CENP-H/K vector was PCR amplified with primers designed for sequence and ligation independent cloning (SLIC) of the PCR fragment into the linearized CENP-I/M vector. The SLIC reaction was then performed to produce a single pFL-based vector with four expression cassettes. Constructs were sequence verified. Baculovirus was then produced and amplified with three rounds of amplification. Expression of CENP-HI57-CKM complex was performed in TnAo38 cells, using a virus:culture ratio of 1:40. Infected cells were incubated for 72 h at 27 °C. Cell pellets were harvested, washed in 1× PBS, and finally resuspended in a buffer containing 50 mM HEPES 7.5, 300 mM NaCl, 1 mM MgCl , 10% glycerol, 5 mM imidazole, 2 mM β-mercaptoethanol, 0.1 mM AEBSF, and 2.5 units per millitre Benzonase (EMD/Millipore). Cells were lysed by sonication, and cleared for 1 h at 100,000g. Cleared cell lysate was then run over a 5 ml Talon superflow column (Clontech) and then washed with 50 mM HEPES 7.5, 1 M NaCl, 10% glycerol, 5 mM imidazole, and 2 mM β-mercaptoethanol. CENP-HI57-CKM complex was eluted with a gradient of 5–300 mM imidazole, and the fractions containing CENP-HI57-CKM pooled, and the His tag cleaved overnight at 4 °C. CENP-HI57-CKM in solution was then adjusted to a salt concentration of 100 mM and a pH of 6.5, before loading on a 6 ml Resource S ion-exchange column (GE Healthcare), equilibrated in 20 mM MES 6.5, 100 mM NaCl, 2 mM β-mercaptoethanol. CENP-HI57-CKM was then eluted with a gradient of 100–1,000 mM NaCl over 20 column volumes, and peak fractions corresponding to CENP-HI57-CKM were pooled and concentrated in a 50 kDa MW Amicon concentrator (Millipore). CENP-HI57-CKM was then loaded onto a Superdex 200 16/600 (GE Healthcare) in 20 mM HEPES 7.5, 150 mM NaCl, 2.5% glycerol, 2 mM TCEP. The sample was concentrated and flash frozen in liquid N before use. CENP-HI57-CKM complex was labelled using the Alexa Fluor 405 C5 Maleimide kit (Thermo Fisher Scientific). A cDNA segment encoding residues 459–561 (the histone fold, HF) of human CENP-T isoform 1, was subcloned in pGEX-6P-2rbs vector as a C-terminal fusion to GST, with an intervening 3C protease site. A cDNA segment encoding human CENP-W was subcloned in the second cassette of the same vector. Similarly, a synthetic cDNA segment encoding human CENP-X isoform 1, codon-optimized for expression in bacteria, was subcloned in pGEX-6P-2rbs vector as a C-terminal fusion to GST, with an intervening 3C protease site. Also, a cDNA segment encoding human CENP-S isoform 1, was subcloned in the second cassette of the same vector. Constructs were sequence-verified. The expression and purification procedure was the same for CENP-T/CENP-W and CENP-S/CENP-X complexes. Escherichia coli BL21 Rosetta cells harbouring vectors expressing GST-CENP-T/CENP-W or GST-CENP-X/CENP-S were grown in Terrific Broth at 37 °C to an absorbance at 600 nm (A ) of 0.6–0.8, then 0.3 mM IPTG was added and the culture was grown at 20 °C overnight. Cell pellets were resuspended in lysis buffer (25 mM Tris/HCl pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT) supplemented with protease inhibitor cocktail (Serva), lysed by sonication, and cleared by centrifugation at 48,000g at 4 °C for 1 h. The cleared lysate was applied to Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) pre-equilibrated in lysis buffer, incubated at 4 °C for 2 h, washed with 70 volumes of lysis buffer and subjected to an overnight cleavage reaction with 3C protease. A heparin column (GE Healthcare) was pre-equilibrated in a mixture of 85% buffer A (20 mM Tris/HCl pH 7.5, 5% glycerol, 1 mM DTT) and 15% buffer B (20 mM Tris/HCl pH 7.5, 2 M NaCl, 5% glycerol, 1 mM DTT). The eluate from glutathione beads was directly loaded onto the heparin column and eluted with a linear gradient of buffer B from 300 to 1,200 mM NaCl in ten bed column volumes. Fractions containing CENP-T(HF)/CENP-W or CENP-S/CENP-X were concentrated in 10-kDa-cut-off Vivaspin concentrators (Sartorius) and loaded onto a Superdex 75 size-exclusion chromatography (SEC) column (GE Healthcare) pre-equilibrated in SEC buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM TCEP). SEC was performed under isocratic conditions at a flow rate of 0.5 ml/min. Fractions containing CENP-T(HF)/CENP-W or CENP-S/CENP-X were concentrated. To form the T(HF)WSX complex, T(HF)W was added to SX at a 1.5 molar excess, incubated for 1 h on ice, and then subjected to separation on a Superdex 200 size-exclusion column to separate tetrameric T(HF)SX complex from T(HF)W dimers. Fractions containing the tetrameric T(HF)WSX complex were then concentrated in a 10-kDa MWCO concentrator to a concentration of 50–250 μM, and flash-frozen. Plasmids for the production of X. laevis H2A, H2B, H3 and H4 histones were a gift from D. Rhodes. X. laevis histone expression and purification, refolding of histone octamers or H2A:H2B dimers, and reconstitution of H3 containing mononucleosomes were performed precisely as described33. Plasmids for the production of the ‘601’ 145-bp DNA were a gift from C. A. Davey. DNA production was performed as described33 with no modifications. For Alexa-647-labelled nucleosomes, the 145-bp DNA fragments (601-Widom) were amplified using fluorescently labelled primers (Sigma-Aldrich). Biotinylated nucleosomes were reconstituted using commercial synthetic 145-bp DNA fragments (601-Widom) (Epicypher). Plasmids for the production of human CENP-A:H4 histone tetramer were a gift of A. F. Straight. Preparations of CENP-A-containing NCPs were performed precisely as described34. For Alexa-647-labelled nucleosomes, the 145-bp DNA fragments (601-Widom) were amplified using fluorescently labelled primers (Sigma-Aldrich, St. Louis, Missouri, USA). Biotinylated nucleosomes were reconstituted using commercial synthetic 145-bp DNA fragments (601-Widom) (Epicypher, Durham, North Carolina, USA). Polycistronic-coexpression plasmid pETDuet–6HisH3.1/CENP-A–H4–6His-H2A–H2B-BFP was generated on the basis of the strategy described previously35 with human histone sequences. The coding sequences of the open reading frames of 6His-H3.1(Ala2–Ile75)/CENP-A(Cys75–Gly140), H4, 6His-H2A1B, and H2B1J-TagBFP were sub-cloned between NcoI and XhoI sites of pETDuet-1 using conventional cloning techniques and the Gibson cloning36. The H3 and CENP-A segments of the chimaera paste within the α1-helix in a structurally seamless manner. One ribosome-binding site was placed upstream of each open reading frame of these four recombinant histones. A TEV protease site was placed between 6His-tag and H3.1/CENP-A-chimaera and a PreScission protease site was placed between 6His-tag and H2A1B to allow tag-removal during protein purification. Protein expression and purification of BFP-labelled H3.1/CENP-A-chimaera histone octamer followed a previous study35 with minor modifications. Purification of the octamer was done according to the previous study35 with minor modifications. After Ni-affinity purification, the octamers were incubated for 15 h at 4 °C with His-TEV protease and His-PreScission protease in buffer A containing 20 mM Tris-HCl pH 8.0, 1.0 M sodium chloride, 1 mM tris(2-carboxyethyl)phosphine (TCEP). The tag-removed octamers were concentrated in buffer B (20 mM Tris-HCl pH 8.0, 2.0 M sodium chloride, 1 mM TCEP) and further purified using Superdex 200 10/300 GL gel-filtration column (GE Healthcare) equilibrated with buffer B. Fractions containing the octamers were pooled, concentrated and stored at −80 °C until used for nucleosome reconstitution. Analytical SEC was performed on a custom-made Superose 6 5/200 in a buffer containing 20 mM HEPES, 300 mM NaCl, 2.5% glycerol, 2 mM TCEP, pH 7.5 on an ÄKTAmicro system. As indicated, the following additional columns were used: Superdex 200 5/150 Increase and Superose 6 5/150. All samples were eluted under isocratic conditions at 4 °C in SEC buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 2.5% glycerol, 2 mM TCEP) at a flow rate of 0.2 ml/min. Elution of proteins was monitored at 280 nm. Fractions (100 μl) were collected and analysed by SDS–PAGE and Coomassie blue staining. To detect the formation of a complex, proteins were mixed at the indicated concentrations in 50 μl, incubated for at least 2 h on ice and then subjected to SEC. Coverslips and glass slides were cleaned by sonication in isopropanol and 1 M KOH or 1% Hellmanex and 70% ethanol, respectively. After functionalization of coverslips with 5% biotinylated poly-l-lysine- PEG for 30 min, flow cells were created with a volume of 10–15 μl. Flow cells were passivated with 1% pluronic F-127 for 1 h and coated with avidin for 30–45 min. After incubation with 10 nM microtubules (10% biotinylated, 10% rhodamine labelled, Cytoskeleton, polymerized according to the manufacturer’s instructions) for 10–20 min, proteins (400 nM) were added in 80 mM Pipes (pH6.8), 125 mM KCl, 1 mM EGTA, 1 mM MgCl and 20 μM Taxol). Flow cells were sealed with wax and imaged with spinning disk confocal microscopy on a 3i Marianas system (Intelligent Imaging Innovations, Göttingen, Germany) equipped with Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany), a CSU-X1 confocal scanner unit (Yokogawa Electric Corporation, Tokyo, Japan), Plan-Apochromat 100×/1.4 numerical aperture DIC oil objective (Zeiss), Orca Flash 4.0 sCMOS Camera (Hamamatsu, Hamamatsu City, Japan) and controlled by Slidebook Software 6.0 (Intelligent Imaging Innovations). Images were acquired as z-sections at 0.27 μm and maximal intensity projections were made with Slidebook Software 6.0 (Intelligent Imaging Innovations). GST pulldown experiments were performed using pre-blocked GSH Sepharose beads in pulldown buffer (10 mM HEPES pH 7.5, 200 mM NaCl, 0.05% Triton, 2.5% glycerol, 1 mM TCEP). GST-CENP-LN as bait at a 1 μM concentration was incubated with NCPs as prey at a 3 μM concentration. The bait was loaded to 12 μl preblocked beads, before the prey was added. At the same time, 1 μg of each protein was added into Laemmli sample loading buffer for the input gel. The reaction volume was topped up to 40 μl with buffer and incubated at 4 °C for 1 h under gentle rotation. Beads were spun down at 500g for 3 min. The supernatant was removed and beads washed twice with 250 μl buffer. Supernatant was removed completely, samples boiled in 15 μl Laemmli sample loading buffer and run on a 14% SDS–PAGE gel. Bands were visualized with Coomassie brilliant blue staining. Preblocking of GSH sepharose beads 750 μl of GSH Sepharose beads were washed twice with 1 ml washing buffer (20 mM HEPES pH 7.5, 200 mM NaCl) and incubated in 1 ml blocking buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 500 μg/μl BSA) overnight at 4 °C rotating. Beads were washed five times with 1 ml washing buffer and resuspended in 500 μl washing buffer to have a 50/50 slurry of beads and buffer. For CENP-C silencing, we used a single siRNA (target sequence: 5′-GGAUCAUCUCAGAAUAGAA-3′; obtained from Sigma-Aldrich), targeting the coding region of endogenous CENP-C mRNA. For an efficient depletion, siRNA for CENP-C was transfected at a concentration of 60 nM for 72 h. For CENP-M silencing, we used a combination of three siRNA duplexes (target sequences: 5′-ACAAAAGGUCUGUGGCUAA-3′; 5′-UUAAGCAGCUGGCGUGUUA-3′; 5′-GUGCUGACUCCAUAAACAU-3′; purchased from Thermo Scientific, Carlsbad, California, USA) targeting the 3′-UTR of endogenous CENP-M. CENP-M siRNA duplexes were used at 20 nM each for 72 h as published3. For CENP-H a single siRNA (target sequence: 5′-CUAGUGUCUCAUGGAUAA-3′ obtained from Dharmacon) targeting the coding region of endogenous CENP-H mRNA was used at 100 nM for 72 h. For CENP-L a single siRNA (target sequence: 5′-UUUAUCAGCCACAAGAUUA-3′ obtained from Dharmacon) targeting the coding region of endogenous CENP-L was used at 100 nM for 72 h. Transfections of RNAi were performed with HyPerFect (QIAGEN) according to the manufacturer’s instructions. Phenotypes were analysed 96 h after first siRNA addition and protein depletion was monitored by western blotting or immunofluorescence. Constructs were created by cDNA subcloning in pcDNA5/FRT/TO-mCherry-IRES vector, a modified version of pcDNA5/FRT/TO vector (Invitrogen). pcDNA5/FRT/TO vector (Invitrogen) is a tetracycline-inducible expression vector designed for use with the Flp-In T-REx system. It carries a hybrid human cytomegalovirus/TetO2 promoter for high-level, tetracycline-regulated expression of the target gene. Parental Flp-In T-REx HeLa cells used to generate stable doxycycline-inducible cell lines were a gift from S. Taylor (University of Manchester, Manchester, UK). They were grown at 37 °C in the presence of 5% CO in Dulbecco’s Modified Eagle’s Medium (DMEM; PAN Biotech) supplemented with 10% TET-free Fetal Bovine Serum (Invitrogen) and 2 mM l-glutamine (PAN- Biotech, 250 μg/ml hygromycin (Invitrogen, Carlsbad, California, USA) and 4 μg/ml blastidicin (Invitrogen, Carlsbad, California, USA). The cell line was regularly tested for mycoplasma contamination. RNAi-depleted cells for various CCAN components were harvested by trypsinization and lysed by incubation in lysis buffer (75 mM HEPES pH 7.5, 150 mM KCl, 1.5 mM EGTA, 1.5 mM MgCl , 10% glycerol, 0.075% NP-40, 90 U/ml benzonase (Sigma)), protease inhibitor cocktail (Serva) at 4 °C for 15 min followed by sonication and centrifugation. Cleared lysate was washed with lysis buffer, resuspended in Laemmli sample buffer, boiled, and analysed by western blotting using 12% NuPAGE gels (Life Technologies). The following antibodies were used: anti-Vinculin (mouse monoclonal, clone hVIN-1; 1:15,000; Sigma-Aldrich, V9131), anti-α-tubulin (mouse monoclonal, Sigma-Aldrich T9026), anti-CENP-C (rabbit polyclonal antibody SI410 raised against residues 23-410 of human CENP-C; 1:1,200; ref. 10), anti-CENP-HK (rabbit polyclonal antibody SI0930 raised against the full length human CENP-HK complex; 1:1,000), anti-CENP-M (rabbit polyclonal antibody raised against the full length human CENP-M), anti-CENP-L (rabbit polyclonal, Acries antibodies 17007-1-AP). Secondary antibodies were affinity-purified anti-mouse (Amersham, part of GE Healthcare), anti-rabbit or anti-mouse (Amersham) conjugated to horseradish peroxidase (1:10,000). After incubation with ECL western blotting system (GE Healthcare), images were acquired with ChemiDocTM MP System (BioRad). Levels were adjusted with ImageJ and Photoshop and images were cropped accordingly. Flp-In T-REx HeLa cells were grown on coverslips pre-coated with 0.01% poly-l-lysine (Sigma). Cells were fixed with PBS/PHEM- paraformaldehyde 4% followed by permeabilization with PBS/PHEM-Triton 0.5%. The following antibodies were used for immunostaining: CREST/anti-centromere antibodies (human auto-immune serum, 1:100; Antibodies, Davis, California), anti-CENP-C (SI410; 1:1,000, or the directly Alexa488 conjugated form of this antibody 1:400), anti-CENP-A mouse monoclonal (Gene Tex GTX13939, 1:500) anti-CENP-HK (SI0930; 1:800 or the Alexa488 directly conjugated form of this antibody 1:800). Rodamine Red-conjugated, DyLight405-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA. Alexa Fluor 647-labelled secondary antibodies were from Invitrogen. Coverslips were mounted with Mowiol mounting media (Calbiochem). All experiments were imaged under identical conditions at room temperature using the spinning disk confocal microscopy of a 3i Marianas system (Intelligent Imaging Innovations, Denver, Colorado, USA) equipped with an Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany), a CSU-X1 confocal scanner unit (Yokogawa Electric Corporation, Tokyo, Japan), Plan-Apochromat 63× or 100×/1.4 numerical aperture objectives (Zeiss) and Orca Flash 4.0 sCMOS Camera (Hamamatsu, Hamamatsu City, Japan) and converted into maximal intensity projections TIFF files for illustrative purposes. Quantification of kinetochore signals was performed on unmodified Z-series images using Imaris 7.3.4 software (Bitplane, Zurich, Switzerland). Z-stacks of single cells were processed in Imaris by creating an ellipsoid of 0.3 μm width and 1 μm height, which was positioned on the CREST signal to cover most of the kinetochore signal in all channels. Four background points with equal ellipsoid size and shape were set between kinetochore dots. Intensity values of single kinetochores were exported in a Microsoft Excel file and the average of the background values was subtracted from every kinetochore value. The mean of all kinetochore signals was taken. For each signal, the mean of the corrected values in mock-depleted cells was set to 1. All other values in perturbation experiments were then normalized to this value to derive the fraction of signal for each measured kinetochore protein compared with control cells. Cross-linking analysis of CENP- ANCP:CHIKLMN:KMN complex or CENP-ANCP:CHIKMNL complex was performed with an equimolar mixture of light and heavy-labelled (deuterated) bis[sulfosuccinimidyl] suberate (BS3-d0/d12, Creative Molecules). The complex was incubated with 0.8 mM BS3 for 30 min at 30 °C and the crosslinking reaction was quenched by adding ammonium bicarbonate to a final concentration of 100 mM. Digestion with lysyl enodpeptidase (Wako) was performed at 35 °C, 6 M urea for 2 h (at enzyme–substrate ratio of 1:50 w/w) and was followed by a second digestion with trypsin (Promega) at 35 °C overnight (also at 1:50 ratio w/w). Digestion was stopped by the addition of 1% (v/v) trifluoroacetic acid (TFA). Cross-linked peptides were enriched on a Superdex Peptide PC 3.2/30 column (300 × 3.2 mm) at a flow rate of 25 μl min−1 and water/acetonitrile/TFA, 75:25:0.1 as a mobile phase. Fractions were analysed by liquid chromatography coupled to tandem mass spectrometry using a hybrid LTQ Orbitrap Elite (Thermo Scientific) instrument. Cross-linked peptides were identified using xQuest11. False discovery rates (FDRs) were estimated by using xProphet12 and results were filtered according to the following parameters: FDR = 0.05, min delta score = 0.85, MS1 tolerance window of −4 to 4 ppm, ld-score >22. The crosslinks were visualized using the webserver xVis ( http://xvis.genzentrum.lmu.de/) (ref. 37). EMSA were performed using either Alexa-647-labelled NCPs, or unlabelled NCPs, at 10 nM. Proteins or protein complexes were added to the nucleosomes at the concentrations indicated and incubated in buffer containing 10 mM HEPES, 150 mM NaCl, 2 mM TCEP, 1% glycerol, 1% Ficoll, 2 mg/ml BSA in 10 μL volume. Samples were then run on 0.75% agarose gel in 0.5× TBE at 4 °C. Gels of unlabelled nucleosomes were stained with SYBRGold (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Gels were imaged using a TyphoonTrio scanner (GE Healthcare, Chicago, Illinois, USA). Quantification was performed using ImageJ, and analysis using Prism (Graphpad, La Jolla, California, USA). CENP-A binding data were fitted with a quadratic binding equation. For CENP-A binding by CHIKMLN, a Hill equation with Hill coefficient of 2.07 was applied, without changes in the apparent K . Sedimentation velocity experiments were performed in an Optima XL-A analytical ultracentrifuge (Beckman Coulter, Palo Alto, California, USA) with Epon charcoal-filled double-sector quartz cells and an An-60 Ti rotor (Beckman Coulter, Palo Alto, California, USA). Samples were centrifuged at 203,000g at 10 °C or 20 °C and 500 radial absorbance scans at either 280 nm or at 497 nm (for samples containing CENP-HI57-CKM complex labelled with Alexa Fluor 488) and collected with a time interval of 1 min. Data were analysed using the SEDFIT software38 in terms of continuous distribution function of sedimentation coefficients (c(s)). The protein partial specific volume was estimated from the amino-acid sequence using the program SEDNTERP. Data were plotted using the program GUSSI in the SEDFIT software38. The GUSSI software is also freely available from http://biophysics.swmed.edu/MBR/software.html. Analysis of NCPs or NCPs bound to CENP-LN was performed at 20 °C in 20 mM HEPES pH 7.5, 10% glycerol, 150 mM NaCl, 1 mM EDTA and 2 mM TCEP (leading to values of buffer density of 1.03503 g/ml and viscosity of 1.002 cP). All other experiments were performed at 10 °C in 10 mM HEPES pH 7.5, 2.5% glycerol and 0.3 M NaCl (leading to values of buffer density 1.02001 g/ml and viscosity of 1.307 cP). To calculate the value of the partial specific volume (V‾, inverse of density) for nucleosomes, we took the value of the 0.55 ml/g for the DNA. This gave a value of V‾ = 0.6565 ml/g for the nucleosomes at 20 °C (or 0.65423 ml/g at 10 °C). The value of the partial specific volume for the CENP-LN bound to CENP-A NCPs is 0.692 ml/g at 20 °C (assuming 2:1 stoichiometry). The value for the CHIKMLN and CENP-A NCPs is 0.71666 ml/g at 10 °C (assuming 2:1 stoichiometry). The value for the HIKM is 0.7394 ml/g at 10 °C and the value for CHIKMLN is 0.73380 ml/g at 10 °C. Biotinylated NCPs (0.5 μM) were incubated with prey proteins (1.5 μM or as indicated) for 30 min on ice in a buffer containing 20 mM HEPES, 200 mM NaCl, 0.05% Triton-X100, 2.5% glycerol, 2 mM TCEP in a reaction volume of 40 μl. Ten microlitres of the protein mix were taken as an input. Ten microlitres of pre-equilibrated streptavidin beads (GE Healthcare, Chicago, IL, USA) were then added to the samples and incubated for 2 min. The samples were then spun down, the supernatant removed, and the beads washed once. Laemmli buffer (1×) was then added to the beads, and heated to 95 °C for 1 min to release all the streptavidin from the beads.


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Human ES cell line H9 (WA-09) and derivatives (SOX10::GFP; SYN::ChR2-EYFP; SYN::EYFP;PHOX2B:GFP;EF1::RFP Ednrb−/−) as well as two independent human iPS cell lines (healthy and familial dysautonomia, Sendai-based, OMSK (Cytotune)) were maintained on mouse embryonic fibroblasts (Global Stem) in knockout serum replacement (KSR; Life Technologies, 10828-028) containing human ES cell medium as described previously7. Cells were subjected to mycoplasma testing at monthly intervals and short tandem repeats (STR) profiled to confirm cell identity at the initiation of the study. Human ES cells were plated on matrigel (BD Biosciences, 354234)-coated dishes (105 cells cm−2) in ES cell medium containing 10 nM FGF2 (R&D Systems, 233-FB-001MG/CF). Differentiation was initiated in KSR medium (knockout DMEM plus 15% KSR (Life Technologies, 10828-028), l-glutamine (Life Technologies, 25030-081), NEAA (Life Technologies, 11140-050)) containing LDN193189 (100 nM, Stemgent) and SB431542 (10 μM, Tocris). The KSR medium was gradually replaced with increasing amounts of N2 medium from day 4 to day 10 as described previously7. For CNC induction, cells were treated with 3 μM CHIR99021 (Tocris Bioscience, 4423) in addition to LDN193189 and SB431542 from day 2 to day 11. ENC differentiation involves additional treatment with retinoic acid (1 μM) from day 6 to day 11. For deriving MNCs, LDN193189 is replaced with BMP4 (10 nM, R&D, 314-BP) and EDN3 (10 nM, American Peptide company, 88-5-10B) from day 6 to day 11 (ref. 3). The differentiated cells are sorted for CD49D at day 11. CNS precursor control cells were generated by treatment with LDN193189 and SB431542 from day 0 to day 11 as previously described7. Throughout the manuscript, day 0 is the day the medium is switched from human ES cell medium to LDN193189 and SB431542 containing medium. Days of differentiation in text and figures refer to the number of days since the pluripotent stage (day 0). For immunofluorescence, the cells were fixed with 4% paraformaldehyde (Affymetrix-USB, 19943) for 20 min, then blocked and permeabilized using 1% bovine serum albumin (BSA) (Thermo Scientific, 23209) and 0.3% Triton X-100 (Sigma, T8787). The cells were then incubated in primary antibody solutions overnight at 4 °C and stained with fluorophore-conjugated secondary antibodies at room temperature for 1 h. The stained cells were then incubated with DAPI (1 ng ml−1, Sigma, D9542-5MG) and washed several times before imaging. For flow cytometry analysis, the cells are dissociated with Accutase (Innovative Cell Technologies, AT104) and fixed and permeabilized using BD Cytofix/Cytoperm (BD Bioscience, 554722) solution, then washed, blocked and permeabilized using BD Perm/Wash buffer (BD Bioscience, 554723) according to manufacturer’s instructions. The cells are then stained with primary (overnight at 4 °C) and secondary (30 min at room temperature) antibodies and analysed using a flow Cytometer (Flowjo software). A list of primary antibodies and working dilutions is provided in Supplementary Table 4. The PHOX2A antibody was provided by J.-F. Brunet (rabbit, 1:800 dilution). Fertilized eggs (from Charles River Farms) were incubated at 37 °C for 50 h before injections. A total of 2 × 105 CD49D-sorted, RFP-labelled NC cells were injected into the intersomitic space of the vagal region of the embryos targeting a region between somite 2 and 6 (HH 14 embryo, 20–25 somite stage). The embryos were collected 36 h later for whole-mount epifluorescence and histological analyses. For RNA sequencing, total RNA was extracted using RNeasy RNA purification kit (Qiagen, 74106). For qRT–PCR assay, total RNA samples were reverse transcribed to cDNA using Superscript II Reverse Transcriptase (Life Technologies, 18064-014). qRT–PCR reactions were set up using QuantiTect SYBR Green PCR mix (Qiagen, 204148). Each data point represents three independent biological replicates. ENC cells from the 11-day induction protocol were aggregated into 3D spheroids (5 million cells per well) in Ultra Low Attachment 6-well culture plates (Fisher Scientific, 3471) and cultured in Neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF). After 4 days of suspension culture, the spheroids are plated on poly-ornithine/laminin/fibronectin (PO/LM/FN)-coated dishes (prepared as described previously26) in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng ml−1, Peprotech, 450-10) and ascorbic acid (100 μM, Sigma, A8960-5G). The ENC precursors migrate out of the plated spheroids and differentiate into neurons in 1–2 weeks. The cells were fixed for immunostaining or collected for gene expression analysis at days 25, 40 and 60 of differentiation. Mesoderm specification is carried out in STEMPRO-34 (Gibco, 10639-011) medium. The ES cells are subjected to activin A treatment (100 ng ml−1, R&D, 338-AC-010) for 24 h followed by BMP4 treatment (10 ng ml−1, R&D, 314-BP) for 4 days9. The cells are then differentiated into SMC progenitors by treatment with PDGF-BB (5 ng ml−1, Peprotech, 100-14B), TGFb3 (5 ng ml−1, R&D systems, 243-B3-200) and 10% FBS. The SMC progenitors are expandable in DMEM supplemented with 10% FBS. The SMC progenitors were plated on PO/LM/FN-coated culture dishes (prepared as described previously26) 3 days before addition of ENC-derived neurons. The neurons were dissociated (using accutase, Innovative Cell Technologies, AT104) at day 30 of differentiation and plated onto the SMC monolayer cultures. The culture is maintained in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng ml−1, Peprotech, 450-10) and ascorbic acid (100 μM, Sigma, A8960-5G). Functional connectivity was assessed at 8–16 weeks of co-culture. SMC-only and SMC-ENC-derived neuron co-cultures were subjected to acetylcholine chloride (50 μM, Sigma, A6625), carbamoylcholine chloride (10 μM, Sigma,C4382) and KCl (55 mM, Fisher Scientific, BP366–500) treatment, 3 months after initiating the co-culture. Optogenetic stimulations were performed using a 450-nm pigtailed diode pumped solid state laser (OEM Laser, PSU-III LED, OEM Laser Systems, Inc.) achieving an illumination between 2 and 4 mW mm−2. The pulse width was 4 ms and stimulation frequencies ranged from 2 to 10 Hz. For the quantification of movement, images were assembled into a stack using Metamorph software and regions with high contrast were identified (labelled yellow in Supplementary Fig. 5). The movement of five representative high-contrast regions per field was automatically traced (Metamorph software). Data are presented in kinetograms as movement in pixels in x and y direction (distance) with respect to the previous frame. We used the previously described method for generation of tissue-engineered colon11. In brief, the donor colon tissue was collected and digested into organoid units using dispase (Life Technologies, 17105-041) and collagenase type 1 (Worthington, CLS-1). The organoid units were then mixed immediately (without any in vitro culture) with CD49D-purified human ES-cell-derived ENC precursors (day 15 of differentiation) and seeded onto biodegradable polyglycolic acid scaffolds (2-mm sheet thickness, 60 mg cm−3 bulk density; porosity >95%, Concordia Fibres) shaped into 2 mm long tubes with poly-l lactide (PLLA) (Durect Corporation). The seeded scaffolds were then placed onto and wrapped in the greater omentum of the adult (>2 months old) NSG mice. Just before the implantation, these mice were irradiated with 350 cGy. The seeded scaffolds were differentiated into colon-like structures inside the omentum for 4 additional weeks before they were surgically removed for tissue analysis. All mouse procedures were performed following NIH guidelines, and were approved by the local Institutional Animal Care and Use Committee (IACUC), the Institutional Biosafety Committee (IBC) as well as the Embryonic Stem Cell Research Committee (ESCRO). We used 3–6-week-old male NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice or 2–3-week-old Ednrbs-l/s-l (SSL/LeJ) mice27 (n = 12, 6 male, 6 female) for these studies. Animal numbers were based on availability of homozygous hosts and on sufficient statistical power to detect large effects between treatment versus control (Ednrbs-l/s-l) as well as for demonstrating robustness of migration behaviour (NSG). Animals were randomly selected for the various treatment models (NSG and Ednrbs-l/s-l) but assuring for equal distribution of male/female ratio in each group (Ednrbs-l/s-l). All in vivo experiments were performed in a blinded manner. Animals were anaesthetized with isoflurane (1%) throughout the procedure, a small abdominal incision was made, abdominal wall musculature lifted and the caecum is exposed and exteriorized. Warm saline is used to keep the caecum moist. Then 20 μl of cell suspension (2–4 million RFP+ CD49D-purified human ES-cell-derived ENC precursors) in 70% Matrigel (BD Biosciences, 354234) in PBS or 20 μl of 70% Matrigel in PBS only (control-grafted animals) were slowly injected into the caecum (targeting the muscle layer) using a 27-gauge needle. Use of 70% matrigel as carrier for cell injection assured that the cells stayed in place after the injection and prevented backflow into the peritoneum. After injection that needle was withdrawn, and a Q-tip was placed over the injection site for 30 s to prevent bleeding. The caecum was returned to the abdominal cavity and the abdominal wall was closed using 4-0 vicryl and a taper needle in an interrupted suture pattern and the skin was closed using sterile wound clips. After wound closure animals were put on paper on top of their bedding and attended until conscious and preferably eating and drinking. The tissue was collected at different time points (ranging from two weeks to four months) after transplantation for histological analysis. Ednrbs-l/s-l mice were immunosuppressed by daily injections of cyclosporine (10 mg kg−1 i.p, Sigma, 30024). The collected colon samples were fixed in 4% paraformaldehyde at 4 °C overnight before imaging. Imaging is performed using Maestro fluorescence imaging system (Cambridge Research and Instrumentation). The tissue samples were incubated in 30% sucrose (Fisher Scientific, BP220-1) solutions at 4 °C for 2 days, and then embedded in OCT (Fisher Scientific, NC9638938) and cryosectioned. The sections were then blocked with 1% BSA (Thermo Scientific, 23209) and permeabilized with 0.3% Triton X-100 (Sigma, T8787). The sections are then stained with primary antibody solution at 4 °C overnight and fluorophore-conjugated secondary antibody solutions at room temperature for 30 min. The stained sectioned were then incubated with DAPI (1 ng ml−1, Sigma, D9542-5MG) and washed several times before they were mounted with Vectashield Mounting Medium (vector, H1200) and imaged using fluorescent (Olympus IX70) or confocal microscopes (Zeiss SP5). Mice are gavaged with 0.3 ml of dye solution containing 6% carmine (Sigma, C1022-5G), 0.5% methylcellulose (Sigma, 274429-5G) and 0.9 NaCl, using a #24 round-tip feeding needle. The needle was held inside the mouse oesophagus for a few seconds after gavage to prevent regurgitation. After 1 h, the stool colour was monitored for gavaged mice every 10 min. For each mouse, total gastrointestinal transit time is between the time of gavage and the time when red stool is observed. The double nickase CRISPR/Cas9 system28 was used to target the EDNRB locus in EF1–RFP H9 human ES cells. Two guide RNAs were designed (using the CRISPR design tool; http://crispr.mit.edu/) to target the coding sequence with PAM targets ~20 base pairs apart (qRNA #1 target specific sequence: 5′-AAGTCTGTGCGGACGCGCCCTGG-3′, RNA #2 target specific sequence: 5′-CCAGATCCGCGACAGGCCGCAGG-3′). The cells were transfected with guide RNA constructs and GFP-fused Cas9-D10A nickase. The GFP-expressing cells were FACS purified 24 h later and plated in low density (150 cells cm−2) on mouse embryonic fibroblasts. The colonies were picked 7 days later and passaged twice before genomic DNA isolation and screening. The targeted region of EDNRB gene was PCR amplified (forward primer: 5′-ACGCCTTCTGGAGCAGGTAG-3′, reverse primer: 5′-GTCAGGCGGGAAGCCTCTCT-3′) and cloned into Zero Blunt TOPO vector (Invitrogen, 450245). To ensure that both alleles (from each ES cell colony) are represented and sequenced, we picked 10 bacterial clones (for each ES cell clone) for plasmid purification and subsequent sequencing. The clones with bi-allelic nonsense mutations were expanded and differentiated for follow-up assays. The ENC cells are plated on PO/LM/FN coated (prepared as described previously26) 96-well or 48-well culture plates (30,000 cm−2). After 24 h, the culture lawn is scratched manually using a pipette tip. The cells are given an additional 24–48 h to migrate into the scratch area and fixed for imaging and quantification. The quantification is based on the percentage of the nuclei that are located in the scratch area after the migration period. The scratch area is defined using a reference well that was fixed immediately after scratching. Migration of cells was quantified using the open source data analysis software KNIME29 (http://knime.org) with the ‘quantification in ROI’ plug-in as described in detail elsewhere30. To quantify proliferation, FACS-purified ENC cells were assayed using CyQUANT NF cell proliferation Assay Kit (Life Technologies, C35006) according to manufacturer’s instructions. In brief, to generate a standard, cells were plated at various densities and stained using the fluorescent DNA binding dye reagent. Total fluorescence intensity was then measured using a plate reader (excitation at 485 nm and emission detection at 530 nm). After determining the linear range, the CD49D+ wild-type and Ednrb−/− ENC precursors were plated (6,000 cell cm−2) and assayed at 0, 24, 48 and 72 h. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. To monitor the viability of wild-type and Ednrb−/− ENC precursors, cells were assayed for lactate dehydrogenase (LDH) activity using CytoTox 96 cytotoxicity assay kit (Promega, G1780). In brief, the cells are plated in 96-well plates at 30,000 cm−2. The supernatant and the cell lysate is collected 24 h later and assayed for LDH activity using a plate reader (490 nm absorbance). Viability is calculated by dividing the LDH signal of the lysate by total LDH signal (from lysate plus supernatant). The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. The chemical compound screening was performed using the Prestwick Chemical Library. The ENC cells were plated in 96-well plates (30,000 cm−2) and scratched manually 24 h before addition of the compounds. The cells were treated with two concentrations of the compounds (10 μM and 1 μM). The plates were fixed 24 h later for total plate imaging. The compounds were scored based on their ability to promote filling of the scratch in 24 h. The compounds that showed toxic effects (based on marked reduction in cell numbers assessed by DAPI staining) were scored 0, compounds with no effects were scored 1, compounds with moderate effects were scored 2, and compounds with strong effects (that resulted in complete filling of the scratch area) were scored 3 and identified as hit compounds. The hits were further validated to ensure reproducibility. The cells were treated with various concentrations of the selected hit compound (pepstatin A) for dose response analysis. The optimal dose (10 μM based on optimal response and viability) was used for follow-up experiments. For the pre-treatment experiments, cells were CD49D purified at day 11 and treated with pepstatin A from day 12 to day 15 followed by transplantation into the colon wall of NSG mice. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. To inhibit BACE2, the ENC precursors were treated with 1 μM β-secretase inhibitor IV (CAS 797035-11-1; Calbiochem). To knockdown BACE2, cells were dissociated using accutase (Innovative Cell Technologies, AT104) and reverse-transfected (using Lipofectamine RNAiMAX-Life Technologies, 13778-150) with an siRNA pool (SMARTpool: ON-TARGETplus BACE2 siRNA, Dharmacon, L-003802-00-0005) or four different individual siRNAs (Dharmacon, LQ-003802-00-0002, 2 nmol). The knockdown was confirmed by qRT–PCR measurement of BACE2 mRNA levels in cells transfected with the BACE2 siRNAs versus the control siRNA pool (ON-TARGETplus Non-targeting Pool, Dharmacon, D-001810-10-05). The transfected cells were scratched 24 h after plating and fixed 48 h later for migration quantification. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. Data are presented as mean ± s.e.m. and were derived from at least three independent experiments. Data on replicates (n) is given in figure legends. Statistical analysis was performed using the Student’s t-test (comparing two groups) or ANOVA with Dunnett test (comparing multiple groups against control). Distribution of the raw data approximated normal distribution (Kolmogorov–Smirnov normality test) for data with sufficient number of replicates to test for normality. Survival analysis was performed using a log-rank (Mantel–Cox) test. Z-scores for primary hits were calculated as Z = (x − μ)/σ, in which x is the migration score value and is 3 for all hit compounds; μ is the mean migration score value, and σ is the standard deviation for all compounds and DMSO controls (n = 224).

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