GSM8605887: hTERT1-RPE1, SMC3, RB KO, M, biol rep 2; Homo sapiens; ChIP-Seq
ChIP assays were carried out on hTERT1-RPE1 cell cultures using approximately 5 million cells per sample and per epitope. Cells were washed once with PBS and fixed with 1.5 mM ethylene glycol bis(succinimidyl succinate) (Thermo Scientific, Cat. # 21565) for 30 min at 25ºC and 1% formaldehyde (Sigma, Cat. # F8775) for 10 min at 37ºC. 20 mM Tris-HCl (pH 7.5) and 150 mM glycine were used to quench cross-linking. Cell pellets were washed twice with ice-cold PBS, and lysates were prepared using 300 μL lysis buffer (50mM Tris-HCl (pH 7.4), 1% SDS, and 0.25% DOC) supplemented with protease and phosphatase inhibitors cocktail (PPI) (Thermo Scientific, Cat. # 78442) for 10 min on ice. Cell lysates were diluted with 900 μL ChIP dilution buffer (50mM Tris-HCl (pH 7.4), 0.1% SDS, 150 mM NaCl, and 1.84% Triton-X) supplemented with PPI. Chromatin was fragmented to a size of 200–700 bases with Branson 250 sonifier. Solubilized chromatin was further diluted with 1800 μL ChIP dilution buffer and immunoprecipitated with the indicated antibodies overnight at 4°C. The primary antibodies were utilized for ChIP: anti-SMC3 (Abcam, Cat. # ab9263, RRID: AB_307122) anti-acSMC3 K105/K106 (Sigma-Aldrich, Cat. # MABE1073), anti-H3K27ac (Active Motif, Cat. # 39034, RRID: AB_2722569), and anti-H3K27me3 (Cell Signaling Technology, Cat. # 12158, RRID: AB_2797834). Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies, Cat. # 10004D) for 2 hours at 4°C and washed 3 times with RIPA 150 Wash Buffer (0.1% SDS, 0.1% DOC 1% Triton X-100, 1 mM EDTA, 10 mM Tris-HCl (pH 8), and 150mM NaCl), 3 times with RIPA 500 Wash Buffer (0.1% SDS, 0.1% DOC, 1% Triton X-100, 1 mM EDTA, 10 mM Tris-HCl (pH 8), and 500 mM NaCl), 3 times with LiCl wash buffer (10 mM Tris-HCl (pH 8), 250 mM LiCl, 0.5% Triton X-100, and 0.5% DOC) and once with ice-cold 10 mM Tris-HCl (pH 8). Chromatin complexes were eluted from the beads using elution buffer (10 mM Tris-HCl (pH 8), 0.1% SDS, 150 mM NaCl, and 5 mM DTT) for 1 hour at 65°C and were treated with RNase A (Roche, Cat. # 11119915001) for 30 min at 37°C. Proteinase K (Thermo Scientific, Cat. # 25530049) treatment and reverse cross-linking was performed for 3 hours at 65°C. After reversal of the crosslinks, immunoprecipitated DNA was extracted with AMP Pure XP beads (Beckman Coulter, Cat. # A63881). ChIP DNA was quantified with Qubit dsDNA HS Assay kit (Thermo Scientific, Cat. # Q32851). 1–2 ng ChIP DNA samples were used to prepare sequencing libraries, using the Ovation Ultralow System V2 kit (TECAN, Cat. # 0344NB-A01).