GSM6602942: AML2_control_H3K27ac; Homo sapiens; ChIP-Seq
Briefly, 20-50 million cells were cross-linked by formaldehyde (1%) and then quenched with 0.125 M glycine. After washing with cold PBS, cells were suspended in lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5%NP-40, 0.25% TritonX-100, and protease inhibitor) and subjected to sonication to shear the chromatin DNA into sizes ranging from 200 to 500 bp. The fragmented chromatin was pre-cleared by centrifugation at 4°C (15,000 rpm, 30 min). The supernatant was incubated with primary antibody and Protein A or G Dynabeads magnetic beads (Invitrogen) at 4°C overnight. The following primary antibodies were used: anti-UTX (Bethyl, A302-374A) and anti-H3K27ac (Abcam, ab4729). After six times wash with RIPA wash buffer (50 mM HEPES-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% Na-Deoxycholate) and once wash with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, and 50 mM NaCl), chromatin was eluted from beads in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). After reverse crosslinking, ChIP DNA was purified using a DNA clean-up kit (Zymo Research). ChIP-seq library was prepared using NEBNext Library Prep Kit following the instructions and samples were sequenced on the NovaSeq 6000 PE150 platform.