Normal Chip H3K23ac BA

After isolation, 1x106 cells were fixed in 1% formaldehyde for 14 minutes and fixation was quenched with the addition of glycine to 125 mM for an additional 1 minute. Cells were washed three times with PBS supplemented with copmlete protease inhibitor cocktail (Roche, 11836145001) and 10 mM sodium butyrate (Sigma Aldrich, 303410), followed by harvesting via scraping from plates. ChIP was performed as previously described{Arrigoni, 2018 #528}, except that no barcode ligation and pooling was performed. Nuclei isolation was performed for 4.5 minutes at peak power 75 Watt, duty factor 2% and 200 cycles/burst using the Covaris E220 instrument (Covaris). Immunoprecipitation (IP) was performed using 2 µg of antibody and protein A/G Dynabeads (Thermo Scientific, 88802). For each IP, an equal amount of chromatin was used per sample. Chromatin quantification was performed by Nanodrop (Thermo Fisher Scientific). The immunoprecipitated DNA was quantified by Qubit 4 Fluorometer (Thermo Scientific) prior to library preparation. Sequencing libraries were prepared using the NEBNext Ultra library preparation kit (New England BioLab, E7645S), and then assessed for quality and quantity via the BioAnalyzer (Agilent). Generated libraries were sequenced on an Illumina NovaSeq instrument (150 bp, paired-end).细胞分离完成后，取1×10⁶个细胞，以1%甲醛固定14分钟，随后加入甘氨酸至终浓度125 mM以终止固定反应，继续孵育1分钟。使用添加了完全蛋白酶抑制剂混合物（罗氏，Roche，货号11836145001）与10 mM丁酸钠（西格玛奥德里奇，Sigma Aldrich，货号303410）的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）洗涤细胞三次，随后通过刮取培养板收集细胞。染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）实验参照此前发表的方法{Arrigoni, 2018 #528}进行，但未开展条形码连接与文库混合步骤。使用Covaris E220仪器（Covaris）进行细胞核分离，参数设置为峰值功率75瓦、占空比2%、每脉冲200个循环，处理时长4.5分钟。免疫沉淀（Immunoprecipitation, IP）反应采用2 μg抗体与蛋白A/G Dynabeads磁珠（赛默飞世尔科技，Thermo Scientific，货号88802）完成，每个IP实验的样品使用等量的染色质。染色质定量通过Nanodrop（赛默飞世尔科技，Thermo Fisher Scientific）完成。文库制备前，采用Qubit 4荧光计（赛默飞世尔科技，Thermo Scientific）对免疫沉淀获得的DNA进行定量。测序文库制备使用NEBNext Ultra文库制备试剂盒（新英格兰生物实验室，New England BioLab，货号E7645S），随后通过BioAnalyzer（安捷伦，Agilent）评估文库的质量与浓度。最终构建完成的文库在Illumina NovaSeq仪器上进行双端测序，读长为150 bp。