rixosom亚基（NOL9，WDR18，PELP1，TEX10），PRC2亚基（EZH2，EED，SUZ12），PRC1亚基（RING1B，PCGF1-4）和CBX1-8 cDNA和人类ES Cell cdna库，并插入PGAD-T7（TakARA）和PGARARA，63042424242424242424242424242424（630443）Y2H分析的质粒。NOL9抗siRNA抗性cDNA是通过PCR产生的。将siRNA靶序列从5'-agacctaagttctgtcgaa-3'突变为5'-CGGCCGAAATTTTGCAGGA-3'，并整合到PCI（Promega，e1731）质粒中以进行分离蛋白的表达。对于细菌蛋白的表达，将cDNA整合到PGEX-6P-1（GE Healthcare，28-9546-48）。

　　用YEPD+腺嘌呤在30°C培养Y2H发芽的酵母菌菌株（Takara）。通过以3,000 rpm离心3分钟来收集酵母细胞OD 0.5。将细胞重悬于0.1 M LIAC（在1x TE缓冲液中）2次。诱饵PGBKT7（0.5μg）表达rixosom，PolyComb和HP1Proteins和Prey PGADT7（0.5μg）载体与10μg载体DNA混合，并将其与10-ML培养物中收集的酵母细胞进一步混合。将DNA酵母混合物与130μl的40％PEG 4000在30°C孵育30分钟。为了转化，加入21μLDMSO并与酵母-DNA混合物充分混合，然后在42°C下进行热休克20分钟。在冰上孵育3分钟后，将细胞通过离心3分钟在4°C下进行沉淀。然后将上清液丢弃，并加入无菌水以重悬于细胞，然后将其铺在双选择性中型SC板上（TRP-，Leu-）在30°C下持续3天。在30°C下，将菌落进一步转移到四个选择性培养基SC板（TRP-，Leu-，His，Ade-）3-4天。为了斑点测定，将细胞在4 ml双选择性SC培养基（TRP-，LEU-）中孵育过夜。然后将细胞稀释至600 nm的光密度，1毫升，将其颗粒颗粒，用灭菌水洗涤一次，重悬于250μl的灭菌水中，并转移到96孔板中。将每个井中的三微升细胞悬浮液铺在双选择性的中型板（TRP-，Leu-）和四核中的中型中级SC板（TRP-，Leu-，His-，Ade-）上四天。

　　HELA（ATCC，CCL-2）和HEK 293FT（Thermofisher，R70007）细胞在含有10％胎牛血清的DMEM中培养。人类胚胎干细胞通过哈佛医学院的基因组编辑和神经退行性的倡议进行了验证，并如前所述进行了培养54。简而言之，用DMEM/F12（含5μgml -1胰岛素和10μgml -1，0.1μgml -1 fgf2，1.7 ng ml -1 tgfβ1，10μgml -1 tgfβ1，10μgml -g ml -1转移）上的0.08 mg ml -1矩阵涂层板上培养细胞。供应商对细胞进行支原体污染测试，为阴性。

　　对于siRNA介导的敲低，使用lipofectamine rnaimax转染试剂（Invitrogen）和siRNA（200 nm）通过遵循制造商的说明来转染细胞。所有siRNA均由佛法合成，并在补充表1中列出。

　　使用Maxiscript T7转录试剂盒（Thermofisher，AM1312）通过体外转录合成小引导RNA。CRISPR – CAS9蛋白通过哈佛医学院细胞生物学系的基因组编辑和神经退行性核心的计划纯化。DNA寡核苷酸模板（由IDT合成，补充表2），引导RNA和CRISPR -CAS9蛋白通过使用霓虹灯转染系统（Thermofisher）的电穿孔传递到细胞中。通过PCR和Miseq测序（Illumina）筛选克隆。

　　将细胞放在带盖玻片的板上。首先用PBS洗涤细胞，并在-20°C下用甲醇固定并用甲醇透化8分钟。然后将细胞与含4％牛血清的PBS中的原代抗体在4°C下孵育4-10小时，然后用二抗抗体染色和1μgml -1 DAPI染色。配备60×/1.40 NA物镜的共聚焦显微镜（Nikon，具有完美焦点和旋转磁盘的Ti）用于图像细胞。NIS元素成像软件用于成像数据收集。图像是用ImageJ（NIH）和Photoshop（Adobe）软件进行后处理的。使用ImageJ评估EZH2和MDN1荧光强度。使用ImageJ直接在视觉上对NPM1灶进行计数。对于MDN1焦点，在对照细胞中的NPM1区域中测量了信号，然后将对照细胞中NPM1染色值最低的焦点用作截止，并且由ImageJ测得的任何具有较高值的​​焦点都计算为MDN1 foci。补充表3中描述了抗体及其来源的列表。

　　为了制备富含染色质的馏分，将细胞用PBS洗涤，然后重悬于冰冷的低音缓冲液中（10 mM HEPES，PH7.9，1.5 mM MGCL2，10 mM KCl，0.2 mM KCl，0.2 mM PMSF，0.2 mm DTT），并在冰上孵育10分钟。然后发散10次，破坏细胞膜。通过以2,000克离心10分钟将细胞核重悬于细胞裂解缓冲液（50 mm HEPES，pH 7.4、150 mM NaCl，1 mM MGCL2，1 mM MM EGTA，1 mM EGTA和0.5％Triton X-100）中，通过管道进行3分钟，以2,000分钟为单位，以10分钟为单位，并以1分钟为单位。将染色质沉淀重悬于IP缓冲液中（50 mM HEPES，pH 7.4、250 mm NaCl，10％甘油，1毫米MGCL2、1 mM EGTA和1％Triton X-100），含有蛋白酶抑制剂鸡尾酒（5056489001，SIGMA）和1 MM MM DNASE I. CHRASE I. CHRASE I. cHROMATIN I.以10,000克离心10分钟。然后将上清液与特定抗体（补充表3）一起孵育，并使用Dynabeads蛋白A/G（热泡）收集免疫复合物。对于银色染色，根据制造商的说明，在5％–20％的BIS-Tris SDS-PAGE凝胶（Biorad）上以5％–20％的BIS-Tris SDS-PAGE凝胶（Biorad）运行样品。对于免疫印迹，将珠子在SDS载荷缓冲液中煮沸5分钟。对于图1F，G，苯并酶（Sigma，E8263）的免疫沉淀，通过将500 U ML -1苯并酶在细胞裂解液中添加500 U ML -1苯并酶进行处理，然后在4°C中孵育1 h，然后与抗体固定珠孵育，然后再孵育。为了进行质谱分析，将蛋白质用0.5 M NH4OH洗脱，并在速度VAC中干燥至完成。

　　对于FLAG – NOL9和FLAG -WDR18免疫沉淀和质谱法，用胰蛋白酶（Promega V5111）在200 mM EPPS缓冲液pH 8.5中消化干蛋白样品。摘要含有2％的乙腈（V/V），并在37°C下进行过夜。直接用TMT10 PLEX试剂（Thermofisher Scientific，90406）标记了摘要。通过质谱检查了标记效率。羟胺淬火（0.3％V/V）持续15分钟后，将反应混合并酸化并通过速度VAC蒸发至接近完成。然后将样品通过碱性反向相色谱法（Thermofisher 84868）分割为12个部分，以10％，12.5％，15％，17.5％，20％，20％，25％，30％，35％，40％，40％，50％，50％，65％，65％和80％乙腈进行洗脱。将馏分汇集成6个馏分（1+7、2+8、3+9、4+10、5+11、6+12），干燥，分阶段倾倒和通过质谱法（Thermo Scientific）上的质谱分析。相对定量遵循多核SPS-MS3方法。在注射之前，使用HPLC使用易于NLC 1200液相色谱系统分离肽，并使用100μm内径毛细管和C18矩阵（2.6μMCACCUCOREC18基质，Thermofisher Scientific）分离肽。用4小时的酸性乙腈梯度分离肽。通过Orbitrap记录（第120,000，质量范围400-1400 TH）测量MS1扫描。碰撞诱导解离（CID）（35％）后，通过离子包装质量分析仪收集MS2光谱。SP（同步前体选择）后，通过高能量碰撞诱导的解离（HCD）（55％）产生TMT报告基因离子，并通过Orbitrap MS3扫描（200 TH）进行定量。使用基于续集（v.28，rev。12）的内部书面软件搜索光谱，并针对人类蛋白质组数据库（Uniprot 07/2014）进行搜索。搜索的质量公差为50 ppm的前体，碎片离子为0.9 da。每个肽允许两次丢失的胰蛋白酶裂解并氧化蛋氨酸 （+15.9949 DA）被动态搜索。对于1％的肽FDR（错误发现率），应用了诱饵数据库策略和线性判别分析（LDA）。折叠蛋白的FDR为1％。通过总和S/N> 200和隔离特异性> 70％，通过求和肽TMT S/N（信号/噪声）来定量蛋白质。最近描述了TMT工作流程和样本准备程序的详细信息55。

　　对于FLAG -PHC2和FLAG -CBX4免疫沉淀和质谱法，我们向珠子中添加了20 µL 8 m尿素，100 mM Epps pH 8.5。我们添加了5 mM TCEP，并在室温下将混合物孵育15分钟。然后，在室温下，我们在室温下添加了10毫米的碘乙酰胺15分钟。我们添加了15 mM DTT来消耗任何未反应的碘乙酰胺。我们添加了180 µL 100 mM EPP pH 8.5。将尿素浓度降低到 <1 M, 1 µg of trypsin, and incubated at 37 °C for 6 h. The solution was acidified with 2% formic acid and the digested peptides were desalted via StageTip, dried via vacuum centrifugation, and reconstituted in 5% acetonitrile, 5% formic acid for LC-MS/MS processing. All label-free mass spectrometry data were collected using a Q Exactive mass spectrometer (Thermo Fisher Scientific) coupled with a Famos Autosampler (LC Packings) and an Accela600 liquid chromatography (LC) pump (Thermo Fisher Scientific). Peptides were separated on a 100 μm inner diameter microcapillary column packed with about 20 cm of Accucore C18 resin (2.6 μm, 150 Å, Thermo Fisher Scientific). For each analysis, we loaded about 2 μg onto the column. Peptides were separated using a 1 h method from 5 to 29% acetonitrile in 0.125% formic acid with a flow rate of about 300 nl min−1. The scan sequence began with an Orbitrap MS1 spectrum with the following parameters: resolution 70,000, scan range 300−1,500 Th, automatic gain control (AGC) target 1 × 105, maximum injection time 250 ms, and centroid spectrum data type. We selected the top twenty precursors for MS2 analysis which consisted of HCD high-energy collision dissociation with the following parameters: resolution 17,500, AGC 1 × 105, maximum injection time 60 ms, isolation window 2 Th, normalized collision energy (NCE) 25, and centroid spectrum data type. The underfill ratio was set at 9%, which corresponds to a 1.5 × 105 intensity threshold. In addition, unassigned and singly charged species were excluded from MS2 analysis and dynamic exclusion was set to automatic. Mass spectrometric data analysis. Mass spectra were processed using a Sequest-based in-house software pipeline. MS spectra were converted to mzXML using a modified version of ReAdW.exe. Database searching included all entries from the S. pombe UniProt database which was concatenated with a reverse database composed of all protein sequences in reversed order. Searches were performed using a 50 ppm precursor ion tolerance. Product ion tolerance was set to 0.03 Th. Carbamidomethylation of cysteine residues (+57.0215 Da) were set as static modifications, while oxidation of methionine residues (+15.9949 Da) was set as a variable modification. Peptide spectral matches (PSMs) were altered to a 1% FDR. PSM filtering was performed using a linear discriminant analysis, as described previously, while considering the following parameters: XCorr, ΔCn, missed cleavages, peptide length, charge state, and precursor mass accuracy. Peptide-spectral matches were identified, quantified, and collapsed to a 1% FDR and then further collapsed to a final protein-level FDR of 1%. Furthermore, protein assembly was guided by principles of parsimony to produce the smallest set of proteins necessary to account for all observed peptides.

　　Proteins for GST pulldown assays were expressed in BL21 Codon Plus Escherichia coli (Agilent Technologies) with 200 μM IPTG induction at 16 °C overnight. Bacteria were then collected and washed with cold PBS, and sonicated (Branson sonicator) for 1 min with 20% amplitude at 4 °C. Sonicated samples were centrifuged at 20,000g for 10 min, and the supernatant was added to 0.5 ml Glutathione Sepharose 4B resin (GE Healthcare, 17075605), which was equilibrated with PBS. GST-tagged proteins were incubated with the resin for 2 h at 4 °C. The resin was then washed 6 times with PBS containing 1% Triton 100. To remove the GST tag, bead-coupled proteins were incubated with PreScission Protease (GE Healthcare, 27-0843-01) in reaction buffer (50 mM Tris-HCl, Ph7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) for 2 h at 4 °C. The GST-tagged PreScission Protease was removed using Glutathione Sepharose 4B resin.

　　For GST pulldown assays, 10 μl 50% slurry of Glutathione Sepharose 4B was used for each sample. GST or GST-tagged proteins (0.1 μM) were incubated with untagged proteins (0.1 μM) in 1 ml PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, Ph7.4) containing 0.5% Triton 100 overnight at 4 °C. Beads were washed 3 times with PBS containing 0.5% Triton 100, resuspended in SDS protein buffer, and boiled for 5 min. Input (2–5%) and bound proteins (10–50%) were run on 4–20% gradient SDS–PAGE gel. SDS–PAGE was performed to separate proteins for 2 h at 80 V, and proteins were transferred to a PVDF membrane (Millipore). The membranes were blocked in 3% milk in PBS with 0.2% Tween-20, and sequentially incubated with primary antibodies and HRP-conjugated secondary antibodies, or directly incubated with HRP-conjugated primary antibodies for chemiluminescence detection. Sources of antibodies can be found in Supplementary Table 3.

　　Flag-tagged proteins were purified from the soluble chromatin fraction using magnetic beads (Sigma, M8823) and eluted with 3×Flag peptides (APExBIO, A6001) in elution buffer (20 mM Hepes-KOH, pH7.5, 100 mM KOAc, 5 mM Mg(OAc)2, 1 mM EDTA, 10% Glycerol). Sucrose gradients (10%-30%) were prepared using the Gradient station (BIOCOMP). An Optima TLX Ultracentifuge equipped with TLS-55 rotor was used for ultracentrifugation for 16 h at 4 °C with 35 k rpm. Gradients of 2.2 ml were fractionated into 22 fractions. One-hundred-microlitre fractions were pipetted from top and protein in fractions was captured using StrataClean resin (Agilent, 400714). Protein samples were boiled in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue) for 3 min at 98 °C, and analysed by immunoblotting following gel electrophoresis (4%–15% precast protein gel with SDS from Biorad, 4561081).

　　Total RNA was extracted using the RNeasy Plus kit (74134, Qiagen) and reverse transcribed into cDNA using gene-specific primers and reverse transcription kit (18090010, ThermoFisher). cDNA was analysed by running PCR on a QuantStudio 7 Flex Real Time PCR System (Applied Biosystem). All reactions were performed using 10 ng RNA in a final volume of 10 μl. PCR parameters were 95 °C for 2 min and 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s, followed by 72 °C for 1 min. All the quantitative PCR data presented were at least three biological replicates. The forward and reverse primers used for RT–qPCR targeted the first exons of the genes. Primer sequences are presented in Supplementary Table 4.

　　Total RNA was isolated from human cells with an RNA purification kit (Qiagen, 74134) and genomic DNA was removed by genomic DNA binding columns in the kit. Two micrograms of total RNA was used for RNA-seq library construction. Poly(A)-containing mRNA was isolated by poly(A) selection beads and further reverse transcribed to cDNA. The resulting cDNA was ligated with adapters, amplified by PCR, and further cleaned to obtain the final library. Libraries were sequenced on an Illumina Hiseq machine (Novogene) to obtain 150 bp paired-ended reads.

　　RNA-seq reads were pseudo aligned using Kallisto 0.45.1. An index was generating using the Ensembl hg19 GTF and cDNA FASTA. Kallisto was run using default parameters with two exceptions: allowing searching for fusions (–fusion) and setting bootstrap to 100 (-b 100).

　　To visualize the mapped RNA-seq with IGV or UCSC genome browser, bam files were generated with Hisat 2.2.0, which was followed by making bigwig files with deeptools (v/3.0.2) (binsize 10). Reads were normalized to reads per genome coverage.

　　Read counts were calculated on a per transcript basis using Kallisto and the above described pseudoalignment. The R package tximport 1.10.1 was used to select the dominant transcript per gene (txOut = FALSE), which was then used for DEseq2 analysis. To analyse only active genes, those with 0 read counts in all samples were removed from the DEseq2 output. As they are not transcribed by PolII, 13 genes on chrM were also removed, resulting in a list of 24,043 active genes. Upregulated genes and downregulated genes are defined with Padj < 0.05 and fold change > 2 or < −2.

　　Aliquots of frozen (−80 °C) permeabilized cells were thawed on ice and pipetted gently to fully resuspend. Aliquots were removed and permeabilized cells were counted using a Luna II, Logos Biosystems instrument. For each sample, 1 million permeabilized cells were used for nuclear run-on, with 50,000 permeabilized Drosophila S2 cells added to each sample for normalization. Nuclear run on assays and library preparation were performed essentially as described56 with modifications noted: 2× nuclear run-on buffer consisted of (10 mM Tris (pH 8), 10 mM MgCl2, 1 mM DTT, 300 mM KCl, 40 μM each biotin-11-NTPs (Perkin Elmer), 0.8 U μl−1 SuperaseIN (Thermo), 1% sarkosyl). Run-on reactions were performed at 37 °C. Adenylated 3′ adapter was prepared using the 5′ DNA adenylation kit (NEB) and ligated using T4 RNA ligase 2, truncated KQ (NEB, per manufacturer’s instructions with 15% PEG-8000 final) and incubated at 16 °C overnight. One-hundred-eighty microlitres of betaine blocking buffer (1.42 g of betaine brought to 10 ml with binding buffer supplemented to 0.6 μM blocking oligonucleotide (TCCGACGATCCCACGTTCCCGTGG/3InvdT/)) was mixed with ligations and incubated 5 min at 65 °C and 2 min on ice prior to addition of streptavidin beads. After T4 polynucleotide kinase (NEB) treatment, beads were washed once each with high salt, low salt, and blocking oligonucleotide wash (0.25× T4 RNA ligase buffer (NEB), 0.3 uM blocking oligonucleotide) solutions and resuspended in 5′ adapter mix (10 pmol 5′ adapter, 30 pmol blocking oligonucleotide, water). 5′ adapter ligation was per Reimer but with 15% PEG-8000 final. Eluted cDNA was amplified with five cycles (NEBNext Ultra II Q5 master mix (NEB) with Illumina TruSeq PCR primers RP-1 and RPI-X) following the manufacturer’s suggested cycling protocol for library construction. A portion of preCR was serially diluted and for test amplification to determine optimal amplification of final libraries. Pooled libraries were sequenced using the Illumina NovaSeq platform.

　　All custom scripts described herein are available on the Adelman Lab Github (https://github.com/AdelmanLab/NIH_scripts). Using a custom script (trim_and_filter_PE.pl), FASTQ read pairs were trimmed to 41 bp per mate, and read pairs with a minimum average base quality score of 20 retained. Read pairs were further trimmed using cutadapt 1.14 to remove adapter sequences and low-quality 3′ bases (–match-read-wildcards -m 20 -q 10). R1 reads, corresponding to RNA 3′ ends, were then aligned to the spiked in Drosophila genome index (dm3) using Bowtie 1.2.2 (-v 2 -p 6–best–un), with those reads not mapping to the spike genome serving as input to the primary genome alignment step (using Bowtie 1.2.2 options -v 2–best). Reads mapping to the hg19 reference genome were then sorted, via samtools 1.3.1 (-n), and subsequently converted to bedGraph format using a custom script (bowtie2stdBedGraph.pl). Because R1 in PRO-seq reveals the position of the RNA 3′ end, the ‘+’ and ‘−’ strands were swapped to generate bedGraphs representing 3′ end position at single nucleotide resolution.

　　For NOL9 KD PRO-seq, we performed 2 sets of PRO-seq experiments, each with two biological replicates. In the first set of experiments, NOL9 depletion resulted in many more upregulated (228) than downregulated (30) genes, while in the second set experiments, nearly the same number of genes were up (162) and down (160) regulated. Furthermore, unlike the first set, in the second set, the extent of overlap between siNOL9 upregulated and downregulated genes with those upregulated in EED-KO or RING1A/B-DKO was similar. Although the basis of this discrepancy is unclear, the correlation between the two biological replicates in Set2 was lower than Set1 raising the possibility that poor growth or inefficient NOL9 depletion in Set2 siNOL9 cells may have resulted in a larger number of non-specifically downregulated genes. We therefore eliminated the Set2 siNOL9 data and used only the 2 biological replicates from the Set1 siNOL9 experiment.

　　To select gene-level features for differential expression analysis, as well as for pairing with PRO-seq data, we assigned a single, dominant TSS and transcription end site (TES) to each active gene. This was accomplished using a custom script, get_gene_annotations.sh (available at https://github.com/AdelmanLab/GeneAnnotationscripts), which uses RNA-seq read abundance and PRO-seq R2 reads (RNA 5′ ends) to identify dominant TSSs, and RNA-seq profiles to define most commonly used TESs. RNA-seq and PRO-seq data from control and siNOL9 cells were used for this analysis, to capture gene activity under both conditions. Exon- and transcript-level features consistent with the resulting TSS to TES windows for 21,004 active genes in HEK 293T cells were selected from an hg19 reference GTF (GRCh38.99 from Ensembl). This filtered list of active genes was used for analyses shown in Figs. 2c–e, 4a–d, Extended Data Figs, 2c, d, 6b–k, as well as for defining differentially expressed genes in PRO-seq data. Differentially expressed genes between control (n = 2) and siNOL9 (n = 2) cells were determined using DESeq2 v1.26.0. Genes were called as differentially expressed using DEseq2’s DESeqDataSetFromMatrix mode at an adjusted P value threshold of <0.05 and fold change >1.5. This revealed 228 genes to be upregulated and 30 genes to be downregulated upon siNOL9.

　　ChIP was performed as previously described with minor modifications57. Cells for ChIP were cultured in 15 cm plates. Cell were first washed with cold PBS, crosslinked at room temperature with 10 mM DMP (ThermoFisher Scientific) for 30 min, and then 1% formaldehyde (ThermoFisher Scientific) for 15 min. Crosslinking reactions were quenched by addition of 125 mM glycine for 5 min. Crosslinked cells were separated by 3 min treatment of 0.05% trypsin (Gibco), and then washed with cold PBS 3 times. In every wash, cells were centrifuged for 3 min at 1,000g at 4 °C. Cell were then resuspended in sonication buffer (pH 7.9, 50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, and 0.5% SDS) and sonicated to shear chromatin into ~300 bp fragments using a Branson sonicator. Sonicated samples were diluted fivefold with ChIP dilution buffer (pH 7.9, 50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate) to obtain a final concentration of 0.1% SDS. Diluted samples were centrifuged at 13,000 rpm for 10 min. The supernatant was pre-cleared with protein A/G or Dynabeads M-280 Streptavidin beads (ThermoFisher) and immunoprecipitated for 3–12 h using 3 μg antibodies and 40 μl protein A/G or Dynabeads M-280 Streptavidin beads. The beads were washed twice with high salt wash buffer A (pH 7.9, 50 mM Hepes, 500 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, and 0.1% SDS), and once with wash buffer B (pH 7.9, 50 mM Hepes, 250 mM LiCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, 0.5% NP-40). The bound chromatin fragments were eluted with elution buffer (pH 8.0, 50 Mm Tris, 10 mM EDTA, 1% SDS) for 5 min at 65 °C. Eluted DNA-proteins complexes were treated with RNase A and crosslinks were reversed overnight at 65 °C. Proteinase K was then added to digest proteins for 1 h at 55 °C. DNA was further purified using PCR Purification Kit (QIAGEN) and analysed by PCR on a QuantStudio 7 Flex Real Time PCR System (Applied Biosystem). PCR parameters were 95 °C for 2 min and 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s, followed by 72 °C for 1 min. All the ChIP–qPCR data presented were at least three biological replicates. Primer sequences are in Supplementary Table 4. Error bars represent standard deviation (three biological replicates).

　　For ChIP–seq, sequencing library was constructed using TruSeq DNA sample Prep Kits (Illumina) and adapter dimers were removed by agarose gels electrophoresis. Sized selected and purified DNA libraries were sequenced on an Illumina Hiseq 2500 machine (Bauer core facility at Harvard University) to obtain 50 bp single-ended reads. ChIP–seq reads were quality controlled with fastqc (v0.11.5) and mapped to the human genome reference (GRCh37/hg19) using bowtie2 (v2.2.9) with default parameters or bowtie (v1.2.2) with parameters -v2 -k1–best. Bam files were generated with samtools 1.3.1, which was followed by making bigwig files with deeptools (v/3.0.2) (binsize 10). Reads were normalized to Reads Per Genome Coverage (RPGC) with deeptools (v/3.0.2) bamCoverage function. To analyse read density at TSS regions, we made heatmaps and metaplots of ChIP–seq samples. TSS was centered in the regions plotted and data were tabulated with the same distance relative to TSS. Matrix files were generated using computematrix function of deeptools (v/3.0.2). based on generated matrix file, heatmaps were generated by PlotHeatmap function, and profiles were generated by plotprofile function or in Prism.

　　To analyse read density and correlation between different ChIP–seq samples, we performed Spearman correlation analysis. Reads density was analysed at all hg19 annotated TSSs (n = 56,335) with multiBigwigSummary function from deeptools (v/3.0.2) to get a npz matrix file. The heatmap Spearman of Pearson correlation was generated by plotCorrelation function of deeptools (v/3.0.2). The heatmaps generated in this study also included all annotated human genes (hg19). The gene list was obtained from https://genome.ucsc.edu. Promoter regions were defined as ±2 kb from TSSs. Peak overlaps were analysed by bedtools (v/3.0.2) intersect function.

　　For co-occupany analysis in Extended Data Fig. 2, peak calling of TEX10, H2AK119ub1, and H3K9me3 was performed with MACS2 (2.1.1.20160309) with Input ChIP–seq sample as control (-p 0.05–broad,–broad-cutoff 0.05, FoldChang>2.5，长度> 800 bp）。

　　为了在图2中定义Tex10结合的目标，使用Homer（版本4.9）称为Tex10峰，并带有风格的组蛋白选项，Sitex10芯片– Seq作为背景。Tex10结合的基因被定义为在TSS±1 Kb区域中具有50次TEX10读数的基因（n = 7,827）；所有其他都被认为是未结合的（n = 13,177）。

　　用于定义图1和图2中的多型靶基因。使用了来自HEK 293FT细胞的2、3，H2AK119UB1芯片seq数据。DeepTools用于计数TSS±2 KB区域中的读数。用k = 2进行k均值聚类。簇O富集H2AK119UB1并将其计为PolyComb靶基因。扩展数据中的Venn图是根据重叠靶基因的数量制作的。DeepTools用于计数TSS±2 KB区域中的读数。k-均值聚类是用k = 3的固定值进行的。群集一被计为靶基因。

　　补充表5列出了本研究中使用的芯片 - 序列数据的来源。

　　对于RNA-seq，Pro-Seq和ChIP-Seq，通过Wilcoxon（未配对）或Mann-Whitney（成对）测试评估了比较的统计显着性。使用的测试和误差条是在每个图中定义的。

　　使用未配对的两尾学生的t检验评估了免疫染色灶的显着性。所有RT – QPCR和芯片-QPCR数据均表示为平均值±S.D。使用GraphPad Prism 8软件。用Microsoft Excel制作了质量规格结果的火山图。

　　有关研究设计的更多信息可在与本文有关的自然研究报告摘要中获得。