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).细胞分离后，取1×10⁶个细胞用1%甲醛固定14分钟，随后加入终浓度125 mM的甘氨酸终止固定反应，继续孵育1分钟。细胞用添加了完整蛋白酶抑制剂混合物（罗氏，11836145001）与10 mM丁酸钠（西格玛奥德里奇，303410）的磷酸盐缓冲液（PBS）洗涤三次，随后通过刮取收集培养板上的细胞。染色质免疫共沉淀（Chromatin Immunoprecipitation, ChIP）实验参照已发表方法[Arrigoni等，2018，#528]进行，仅不进行条形码连接与混合步骤。使用Covaris E220超声仪（Covaris）进行细胞核分离，参数设置为峰值功率75 W、占空比2%、每个脉冲200个循环，处理时长4.5分钟。免疫沉淀（Immunoprecipitation, IP）实验使用2 μg抗体与蛋白A/G磁珠（赛默飞世尔科技，88802）完成。每份免疫沉淀反应均使用等量的染色质样本。采用Nanodrop分光光度计（赛默飞世尔科技）对染色质进行定量。在文库制备前，采用Qubit 4荧光计（赛默飞世尔科技）对免疫沉淀得到的DNA进行定量。测序文库使用NEBNext Ultra文库制备试剂盒（新英格兰生物实验室，E7645S）构建，随后通过安捷伦BioAnalyzer生物分析仪评估文库的质量与浓度。构建完成的文库在Illumina NovaSeq测序仪上进行双端150 bp测序。