ChIP-Seq

The esophageal cancer cells were treated with formaldehyde to covalently crosslink proteins to DNA. Then sonication was performed to cut the chromatin into fragments of 100–300 bp. Immunoprecipitation with an antibody against acetylated histone H3K9 was carried out to enrich for acetylated histone H3-bound DNA compared to total chromatin. Then next generation sequencing was performed using Zhongkangbo Biotechnology (HiSeq 2500, Beijing, China). Briefly, independent DNA libraries were produced with the Illumina TrueSeq Library Kit (San Diego, CA, USA) as per manufacturer’s instructions, and libraries were sequenced on the Illumina HiSeq producing 50 bp single end reads. Then Bowtie2 (John Hopkins University (Baltimore, MD, USA) was used to align reads to the human genome (GRCh37/hg19), and peaks were called for each biological replicate using Model-based Analysis of ChIP-Seq (MACS2).