GSM4404007: DMSO-H3K27me3; Homo sapiens; ChIP-Seq
Cells were cross-linked in 1% formaldehyde for 10 min at room temperature (RT) with gentle swirling, and crosslinking was stopped by adding glycine to a final concentration of 0.125 M and incubating at RT for 5 min. Cells were harvested and an aliquot of 1 × 107 cells were used per ChIP reaction. Each cell aliquot was resuspended in 0.5 mL of RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS, pH 7.4), and chromatin was fragmented on ice to 200 to 500 bp in size with 5 pulses of 30 sec at 50% power with a Sonics VibraCell sonicator. 2 μg primary antibodies as listed in Supplementary Table S4 or control IgG were pre-conjugated with Protein G magnetic beads (Pierce) at 4°C overnight. 1% of sheared chromatin was saved as input, and the rest was incubated with pre-conjugated antibody on a rotator at 4°C overnight. The beads were washed 5 times with LiCl wash buffer (0.1 M Tris, 0.5 M LiCl, 1% NP-40, 1% sodium deoxycholate, pH 7.5) followed by a single wash with TE buffer (10 mM Tris-HCl, 0.1 mM Na2EDTA, pH 7.5). The beads were then resuspended in 200 μL IP elution buffer (1% SDS, 0.1 M NaHCO3) and incubated at 65°C for 1 hour in a heat block with vortexing. The supernant was collected and together with input was incubated at 65°C for 16 hours to reverse cross-links. DNA was purified with QIAquick PCR Purification Kit (Qiagen) and eluted in 50 μL EB. ChIP's DNA was subjected to end-repair and then was 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated fragments. Fragments were amplified by PCR and PCR products were purified and selected with the Agencourt AMPure XP-Medium kit. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. Library was qualified by Qubit ssDNA kit.
GEO
GSM4404007