Quantifying Histone Modifications by ChIP-seq

Chromatin immunoprecipitation (ChIP) was performed as described previously^{40 (link)}. Cells were transfected with siRNAs as described above and incubated for 72 h. Aliquots of cells (1 × 10^{6 (link)}) were then fixed and crosslinked, after which samples were immunoprecipitated for 12 h at 4 °C using a rabbit anti-acetylated histone H3 lysine 27 (H3K27ac) pAb (#39,133; Active Motif, Carlsbad, CA, USA), a rabbit anti-trimethylated histone H3 lysine 4 (H3K4me3) mAb (#04–745; Merck, Darmstadt, Germany), or normal mouse IgG (#140–09,511, Fujifilm, Tokyo, Japan), which served as a control. For ChIP-seq analysis, samples were sequenced using an Ion Proton System (Thermo Fisher Scientific). Sequencing data were mapped to the human genome hg19 using bowtie2, after which peaks were called using MACS2 and were annotated using HOMER v4.11. ChIP-seq results were visualized using Microsoft Excel, Java TreeView and Integrative Genomics Viewer. ChIP-qPCR was carried out using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). The sequences of the PCR primers are listed in Supplementary Table S2. The Gene Expression Omnibus accession number for the microarray data is GSE178613.