ChIP-Seq Analysis of H3K27ac in NB4 Cells

3–5 × 10⁷ cells were crosslinked with 1% formaldehyde for 10 min and neutralized with 1.25 M glycine for 5 min at room temperature. Fixed cells were harvested, lysed, and sonicated using a Bioruptor (Diagenode, Liège, Belgium). Sonicated chromatin was incubated with 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). Samples were sequenced on a novaseq 6000 platform (Novogene Bioinformatics Technology Co., Ltd. Beijing, China). Raw data of ChIP-Seq H3K27ac analysis for NB4 cell line was aligned to the reference genome (UCSC hg38) using Bowtie2 (v 2.3.5) [34 (link)], with alignment parameters -p 4 -q -x. Peaks were identified using MACS2 (v2.0.9) [35 (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 then bigwig files were visualized by Integrative Genomics Viewer (IGV) [36 (link)].