GSM4979114: POLR3A_monocyte_1; Homo sapiens; ChIP-Seq
Equal numbers of THP-1 monocytes and THP-1 derived macrophages were collected (~10 million cells per ChIP experiment) and resuspended in growth media at 1 x 106 cells/mL and cross-linked with rotation at room temperature in 1% formaldehyde for 10 minutes. Cross-linking was quenched with the addition of 200 mM glycine and an additional 5 minutes of rotation at room temperature. Cross-linked cells were then spun down and resuspended in 1x RIPA lysis buffer, followed by chromatin shearing via sonication (3 cycles using a Branson sonicator: 30 seconds on, 60 seconds off; 15 additional cycles on a Bioruptor sonicator: 30 seconds on, 30 seconds off). Individual ChIP experiments were performed on pre-cleared chromatin using antibody-coupled Dynabead protein G (ThermoFisher) magnetic beads. Anti-histone H3 (acetyl K27) antibody was obtained from Abcam (ab4729), POLR3D antibody was obtained from abcam (ab86786; Lot# GR267691-1). 3-5 ug of antibody per ChIP was coupled to 18 uL beads and rotated overnight with sheared chromatin at 40 C. Beads were then washed 5x in ChIP wash buffer (Santa Cruz), 1x in TE, and chromatin eluted in TE + 1% SDS. Cross-linking was then reversed by incubation at 650 C overnight, followed by digestion of RNA (30 min RNase incubation at 370C) and digestion of protein (30 min proteinase K incubation at 450C). ChIP DNA was then purified on a minElute column (Qiagen) libraries were prepared for sequencing using standard Illumina protocols