GSM7593120: KAT6A_MGD1149_h3k23ac; Homo sapiens; ChIP-Seq
The dermal fibroblast cells were grown to 80-90% confluency and then fixed using fresh media containing 1% formaldehyde. Cells were incubated in formaldehyde media for 10 minutes, and then the reaction was neutralized with 1.25M glycine. After this, the media was removed and cells were washed twice with ice cold PBS containing protease inhibitor. Post PBS wash, the cells were scraped and collected in a 1.5 ml tube. The cell pellets were resuspended in ChipLysis buffer and sonication was performed using Diagenode bioruptor at high for 45 cycles (30 sec on, 30 sec off). 5 ul of sonicated product was de-crosslinked and purified to check optimal lysis (200-600 bp). The sonicated chromatin was pre-cleared with Antigen G dynabeads for 2 hours on a nutator at 4 0 C. The pre-cleared chromatin was then incubated with 5 ul of H3K9ac (Cell signaling#9649), 2 ul of H3K23ac (AbCam #ab177275), and 4ul of IGg (cell signaling #2729) at 4 0C overnight. The next day, the antibody-chromatin complex was incubated with antigen G dynabeads for an additional 2 hours on a nutator, and then the beads were washed by a series of buffers and then de-crosslinked at 65 0C overnight. Post de-crosslinking, the DNA was purified and processed for library preparation using illumina Chip library prep (illumina#15034288) was used for library construction. Chip libraries were sequenced on illumina Hiseq3000 using single read 75 bp read-length.