GSM3356982: N20021007 H3K27ac; Homo sapiens; ChIP-Seq
For primary tissues, fresh-frozen cancer and normal tissues were dissected using a razor blade in liquid nitrogen to obtain ~5mg sized piece for each ChIP. Tissue pieces were fixed in 1% formaldehyde/PBS buffer for 10 min at room temperature. Fixation was stopped by addition of glycine to a final concentration of 125 mM. Tissue pieces were washed 3 times with TBSE buffer. For cell lines, 1 million fresh harvested cells were fixed in 1% formaldehyde/medium buffer for 10 minutes (min) at room temperature. Fixation was stopped by addition of glycine to a final concentration of 125 mM. Fixed cells were washed 3 times with TBSE buffer, and centrifuged (5,000 r.p.m., 5 min). Pelleted cells and pulverized tissues were lysed in 100 µl 1% SDS lysis buffer and sonicated to 300-500bp using a Bioruptor (Diagenode). ChIP was performed using antibodies: to H3K27ac (ab4729, Abcam). After recovery of ChIP and input DNA, whole-genome-amplification was performed using the WGA4 kit (Sigma-Aldrich) and BpmI-WGA primers. Amplified DNAs were purified using PCR purification columns (QIAGEN) and digested with BpmI (New England Biolabs) to remove WGA adapters. 30ng of amplified DNA was used for each sequencing library preparation (New England Biolabs).