Normal ChIP H3K9ac Control Primary human hepatocytes

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。用添加了完全蛋白酶抑制剂混合物（Roche，货号11836145001）和10 mM丁酸钠的磷酸盐缓冲液（PBS）洗涤细胞三次，随后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）实验按照此前报道的方法（Arrigoni等，2018，文献编号#528）进行，仅不进行条形码连接与混合步骤。使用Covaris E220仪器（Covaris）进行细胞核分离，设置参数为：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）实验使用2 μg抗体与蛋白A/G磁珠（Thermo Scientific，货号88802）完成。每次免疫沉淀实验中，每个样本使用等量的染色质。采用Nanodrop（Thermo Fisher Scientific）对染色质进行定量。在文库制备前，使用Qubit 4荧光定量仪（Thermo Scientific）对免疫沉淀得到的DNA进行定量。使用NEBNext Ultra文库制备试剂盒（New England BioLab，货号E7645S）构建测序文库，随后通过BioAnalyzer（Agilent）评估文库的质量与浓度。构建得到的文库在Illumina NovaSeq仪器上进行测序，测序模式为150 bp双端测序。