ChIP-seq of H3K4me3 Enrichment

ChIP using antibodies against trimethyl H3K4me3 (Millipore) was carried out according to a previously described method [49 (link)]. Briefly, cultured cells were cross-linked in 1% formaldehyde for 10 min at 37 °C. After the addition of 1/10 volume of 1.25 M glycine and incubation for 5 min, fixed cells were washed twice with cold PBS buffer. Soluble chromatin was prepared by sonication (Bioruptor sonicator; Cosmo Bio) to an average DNA size of 500 bp in sonication buffer and immunoprecipitated in IP buffer (20 mM Tris–HCl, pH 8.0, 600 mM NaCl, 1 mM EDTA, 0.05% SDS, 1.0% Triton X-100, 20% glycerol, 1.5 μM aprotinin, 10 μM leupeptin, 1 mM DTT and 40 μM MG132). Protein G sepharose (Amersham, USA) blocked with BSA was added and the antibody–chromatin complex recovered by centrifugation. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad). Primers for alphoid^tetO repeat (tetO) as well as for a control 5S ribosomal DNA, are listed in Supplementary Table S1. At least three independent ChIP experiments were performed to estimate the level of enrichment.