GSM5593695: TS516_R2_H3K4me3_ChIPseq; Homo sapiens; ChIP-Seq
20 x 106 cells (TF-1 and NHA cells) or 1 x 106 cells (MCTS lines) were collected per replicate, cross-linked with 1% formaldehyde for 10 minutes at room temperature on a rocker, and quenched with 140mM glycine for 5 minutes at room temperature. Cells were washed 3 times with PBS and snap frozen. Cell pellets were resuspended in ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH8.1) containing protease inhibitor cocktail (Roche). Chromatin was sonicated using a Sonic Dismembrator Model 100 (Fisher Scientific) with rounds of 10 seconds on, 30 seconds off at 30% power (TF1 cells: 11 rounds, NHA cells: 9 rounds, MCTS lines: 7 rounds). Shearing efficacy was confirmed by agarose gel electrophoresis. Immunoprecipitations were performed with the equivalent of 1 x 106 cells for each sample using the Magna ChIP A/G kit (Millipore) and rabbit monoclonal H3K4me3 antibody (Millipore, 15-10C-E4) or normal rabbit IgG (Millipore, #12-370). Drosophila spike-in chromatin (Active Motif) was added along with Drosophila anti-H2Av (Active Motif) to samples prior to immunoprecipitation. Chromatin was incubated with primary antibodies overnight at 4 °C with constant agitation. Inputs were prepared from sonicated controls for each cell line. Sequencing libraries were generated from purified IP sample DNA using Swift 2S ligation chemistry