ChIP-seq Library Preparation Protocol

The preparation of ChIP and input DNA libraries was performed as previously described [32 (link)]. In brief, two cells were crosslinked with 1% formaldehyde for 5 min at room temperature and quenched with glycine (125 mM). Cells were then put on ice, resuspended in cold cell lysis buffer [140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% SDS, and protease inhibitors (Roche)]. Nuclei were sonicated into fragments of 200–1000 bp in size. The chromatin fragments were precleared and then immunoprecipitated with Protein A + G magnetic beads coupled with anti-H3K4me3 (ab8580; Abcam), anti-H3K27ac (ab4729; Abcam), anti-H3K27me3 (07-449; Millipore), and anti-CTCF (ab70303; Abcam). After reverse crosslinking, immunoprecipitated DNA and input DNA were end-repaired and adapters were ligated to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module (E7442; NEB) and NEBNext Ultra Ligation Module (E7445; NEB). High-throughput sequencing of the ChIP fragments was performed using Illumina NextSeq 500, following the manufacturer’s protocol.