ChIP-seq Analysis of Histone H3K27 Acetylation

Furthermore, 3-5 × 10⁷ cells were cross-linked with 1% formaldehyde for 10 minutes and neutralized with 1.25 M glycine at room temperature for 5 minutes. Bioruptor (Diagenode, Liège, Belgium) was used to collect, lyse, and sonicate fixed cells. Sonicated chromatin was incubated with an anti-histone H3 (acetyl K27) antibody (cat. No. ab4729; Abcam, Cambridge, UK) overnight at 4°C. DNA was eluted and purified using a QIAquick PCR purification kit (cat. No. 208106; Qiagen, Hilden, Germany). The samples were sequenced on the NovaSeq 6000 platform (Novogene Bioinformatics Technology Co., Ltd. Beijing, China) and a BGISEQ 2000 platform (Beijing Genomics Institute, Shenzhen, China). Raw data of ChIP-seq H3K27ac analysis were aligned to the reference genome (UCSC hg38) using Bowtie2 (v 2.3.5) [17 (link)], with alignment parameters -p 4 -q -x. Peaks were identified using MACS2 (v2.0.9) [18 (link)], with parameters -g hs -n test -B -q 0.01. The bedGraph files generated by MACS2 were converted to bigwig files using the UCSC bedGraphToBigWig tool, and bigwig files were then visualized by Integrative Genomics Viewer (IGV) [19 (link)]. Superenhancers were then identified using the ROSE (Rank Order of Superenhancers) method [20 (link), 21 (link)], with parameters -s 12500 -t 2000.