GSM8545893: COPD_115_K4me3; Homo sapiens; ChIP-Seq
2 milions  neutrophils were crosslinked with 1 % formaldehyde for 10 min. Cross-linking reaction was stopped adding 125mM glycine dor 5 min.  After fixation, cells were washed with ice-cold PBS, pelleted by centrifugation and resuspend in 900 ml L1 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 500 ml L2 buffer (50 mM Tris, pH 8.0, 1 % SDS, 5 mM EDTA) supplemented with protease inhibitors. Chromatin was sheared  using a BANDELIN SONOPLUS ultrasonic homogenizers HD 2070 (Bandelin, Berlin, Germany).  Immunoprecipitations were carried out overnight at 4°C using 3ml antibody. Immune complexes were then collected by adding 15 µl of protein A magnetic beads (Thermo Fisher) for 20min at 4°C under gentle rotation. Beads werewashed 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. 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. Prior to lybrary preparation, ChIP-DNA was cleaned and concentrated using the ChIP DNA Clean & Concentrator kit (Zymo Research). ChIP DNA libraries were prepared using the NEXTflex Rapid DNA Sequencinf Bundle (BIOO SCIENTIFIC) according to manufacturer protocol.