GSM8323365: monocytes,H3K27ac,NT,HD2,ChIP; Homo sapiens; ChIP-Seq
Protein-DNA cross-linking was achieved by incubating 2.5x106 cells with 1 % formaldehyde for 10 min at room T, under gentle agitation. Cross-linking reaction was stopped by adding glycine to a final concentration of 125 mM, and incubating cells at room T for five more minutes. After fixation, cells were washed with ice-cold PBS, collected by scraping, and finally pelleted by centrifugation (5 min, 300xg, 4°C). Pellets were suspended in 900 µl L1 lysis buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 0.1 % IGEPAL, 10 % glycerol) containing protease inhibitors. Nuclei were pelleted at 1000xg at 4°C and resuspended in 300 µl L2 lysis buffer (50 mM Tris, pH 8.0, 1 % SDS, 5 mM EDTA) including protease inhibitors. Chromatin was sheared to an average DNA size of 300-400 bp by sonication on wet ice [6 pulses of 15 seconds at the 50 % maximum potency, with 15 second pauses, using a BANDELIN SONOPLUS ultrasonic homogenizers HD 2070 (Bandelin, Berlin, Germany)]. Lysates were then cleared by centrifugation to remove debris (10 min, 13000xg, 12°C), and diluted 10x in dilution buffer (50 mM Tris, 5 mM EDTA, 200 mM, 0.5 % IGEPAL). Immunoprecipitations were carried out overnight at 4°C using 5 µg/ml antibodies. Immune complexes were then collected by adding 15 µl of protein A sepharose-coupled magnetics beads (GE Healthcare, Piscataway, NJ, USA) for 1 h at 4°C under gentle rotation. Beads were then immobilized on a magnetic support and washed three times in washing buffer (20 mM Tris, pH 8.0, 0.1 % SDS, 2 mM EDTA, 1% IGEPAL, 500 mM NaCl) and once in TE. The resulting protein complexes were then eluted in TE containing 2 % SDS, and reversed crosslinked by an overnight incubation at 65°C. The DNA was purified by QiaQuick PCR purification kit (Qiagen) according to the manufacturer's instructions, and eluted in 50-100 µl. ChIP DNA was prepared for NextSeq 500 sequencing following TruSeq DNA sample preparation guide (Illumina, Cambridge, UK). In brief, 10-50 ng purified DNA from chromatin immunoprecipitation were adapter-ligated and PCR-amplified according to the manufacturer's protocol (Illumina). Sequencing libraries were multiplexed and ran on NextSeq 500 sequencer (Illumina).