ChIP-seq Protocol for Murine Embryonic Stem Cells

ChIP assays were performed from 10⁷ mESC per experiment, according to previously described protocol with slight modification (Rada-Iglesias et al., 2011 (link)). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched by glycine at a final concentration of 0.125 M. Chromatin was sonicated to an average size of 0.5–2 kb, using Bioruptor (Diagenode). A total of 5 μg of antibody was added to the sonicated chromatin and incubated overnight at 4°C. Subsequently, 50 μl of protein G Dynal magnetic beads were added to the ChIP reactions and incubated for ~4 hr at 4°C. Magnetic beads were washed and chromatin eluted, followed by reversal of crosslinks and DNA purification. ChIP DNA was dissolved in water. ChIP-seq and input libraries were prepared according to Illumina protocol and sequenced using Illumina Genome Analyzer. Following library preparation, samples were pooled and sequenced on an Illumina NextSeq instrument using 76 base-pair single-end reads on a NextSeq high output kit (Illumina).