ChIP-seq Protocol for Chromatin Immunoprecipitation

ChIP was carried out according to a previously described method (31 (link)). Cultured cells were cross-linked in 1% formaldehyde for 10 min at 37°C. After addition of 1/10 volume of 1.25 M glycine and incubation for 5 min, fixed cells were washed twice with cold phosphate buffered saline (PBS) buffer. Soluble chromatin was prepared by sonication in water bath-based sonicator (Bioruptor sonicator; Cosmo Bio) to an average DNA size of 500 bp and immnnoprecipitated in IP buffer (0.01% sodium dodecyl sulphate (SDS), 1.1% Triton X- 100, 1.2 mM ethylenediaminetetraacetic acid, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). Protein A agarose/salmon Sperm DNA (Millipore, #16–157) was added, and the antibody-chromatin complex was recovered by centrifugation. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a 7900HT Fast real-time PCR detection system (Applied Biosystems) and SYBR Green PCR Master Mix (Applied Biosystems, #4309155). Primers for HORs regions of chromosome 21 and chromosome X, tetO-2 mer of the alphoid^tetO DNA array, HORs D5Z1 and D5Z2 regions of chromosome 5 were described previously (30 (link),32 (link)) (see also Supplementary Table S1).