Normal Chip H4K16ac 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).分离完成后，取1×10⁶个细胞，用1%甲醛固定14分钟；随后加入终浓度125mM的甘氨酸终止固定反应，继续孵育1分钟。用添加了完整蛋白酶抑制剂混合物（Roche，货号11836145001）与10mM丁酸钠（Sigma Aldrich，货号303410）的PBS洗涤细胞三次，随后通过刮取收集培养板中的细胞。染色质免疫共沉淀（Chromatin Immunoprecipitation，ChIP）实验参照已发表方案（Arrigoni等，2018，文献编号#528）进行，仅不执行条形码连接与样本混合步骤。使用Covaris E220仪器（Covaris）进行细胞核分离：峰值功率75W，占空比2%，每爆发200个循环，处理时长4.5分钟。免疫沉淀（Immunoprecipitation，IP）反应使用2μg抗体与Protein A/G磁珠（Thermo Scientific，货号88802）进行，所有IP样本均使用等量染色质。采用Nanodrop分光光度计（Thermo Fisher Scientific）对染色质进行定量；文库制备前，使用Qubit 4荧光计（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，随后通过生物分析仪（Agilent，BioAnalyzer）评估文库的质量与浓度。最终构建的文库在Illumina NovaSeq仪器上进行150bp双端测序。