Normal Chip H3K23ac Control

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。使用添加了完全蛋白酶抑制剂混合物（Roche，货号11836145001）与10 mM丁酸钠（Sigma Aldrich，货号303410）的磷酸盐缓冲液（Phosphate Buffered Saline，PBS）洗涤细胞三次，再通过刮取的方式从培养板中收集细胞。染色质免疫共沉淀（Chromatin Immunoprecipitation，ChIP）实验参照已发表方法[Arrigoni等，2018，编号528]开展，仅省略了条码连接与文库合并步骤。使用Covaris E220仪器（Covaris）进行细胞核分离，设置峰值功率75 W、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）实验采用2 μg抗体与蛋白A/G磁珠（Thermo Scientific，货号88802）完成，每组免疫沉淀实验均使用等量的染色质样品。染色质浓度通过超微量紫外分光光度计（Nanodrop，Thermo Fisher Scientific）测定。在构建测序文库前，采用Qubit 4荧光定量仪（Thermo Scientific）对免疫沉淀得到的DNA进行定量。测序文库采用NEBNext Ultra文库构建试剂盒（New England BioLab，货号E7645S）制备，随后通过生物分析仪（Agilent）评估文库的质量与浓度。最终构建的文库在Illumina NovaSeq测序仪上进行双端150 bp测序。