GSM5026287: 22_time60_H3K9me3; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed as previously described (Kanki et al., 2011; Kanki et al., 2017). In brief, 10 million HUVECs were cross-linked with 1% formaldehyde (Wako) for 10 min. For p65 and FLAG ChIP analysis, cells were fixed with 2 mM EGS (ethylene glycol bis [succinimidyl succinate]) (Thermo Fisher Scientific) for 1 hr prior to formaldehyde fixation. After neutralization by using 0.2 M glycine, cells were collected, re-suspended in 2 ml SDS lysis buffer composed of 10 mM Tris-HCl; pH8.0 (Thermo Fisher Scientific), 150 mM NaCl (Thermo Fisher Scientific), 1% SDS (Sigma-Aldrich), 1mM EDTA; pH 8.0 (Thermo Fisher Scientific), cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich), and fragmented by the Picoruptor (40 cycles for histones and p65, and 20 cycles for FLAG, 30 sec on/30 sec off; Diagenode, Liege Science Park, Belgium). Sonicated solution was diluted by ChIP dilution buffer (20 mM Tris-HCl; pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (Sigma-Aldrich)) up to 10.3 ml, which was used for immunoprecipitation (10 ml) and the rest 300 μl as a non-immunoprecipitated chromatin (INPUT). Specific antibody was bound with magnetic Dynabeads M-280 and applied to diluted sonicated solution for immunoprecipitation. Antibodies against H3K4me3, H3K27ac, H3K9me2, H3K27me3 (MAB Institute, Inc. Hokkaido, Japan), p65 (abcam for ChIP-Seq and Santa Cruz Biotechnology Inc for ChIP-PCR), and FLAG (Sigma-Aldrich) were used. Prepared DNA was quantified using NanoDrop 2000 (Thermo Fisher Scientific) and more than 10 ng of DNA was processed for qPCR or sequence. Libraries were prepared according to Illumina's instructions.