title: GSM6166423: T47D_dCDK_FLAG; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP assays were carried out on RPE1, BJ, MCF10A and T47D 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. No. 21565) for 30 min at 25oC and 1% formaldehyde (Sigma, Cat. No. F8775) for 10 min at 37oC. 20mM Tris-HCl pH7.5 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, 0.25% DOC) supplement with protease and phosphatase inhibitors cocktail (PPI) (Thermo Scientific, Cat. No. 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, 150mM NaCl, 1.84% Triton-X) supplement with PPI and 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. Anti-DYKDDDDK Tag (Cell Signaling, Cat. No. 14793) was used for FLAG-RB ChIP. Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies, Cat. No. 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, 1mM EDTA, 10mM Tris-HCl pH 8, 150mM NaCl), 3 times with RIPA 500 Wash Buffer (0.1% SDS, 0.1% DOC 1% Triton X-100, 1mM EDTA, 10mM Tris-HCl pH 8, 500mM NaCl), 3 times with LiCl wash buffer (10mM Tris-HCl, pH 8, 250mM LiCl, 0.5% Triton X-100, 0.5% DOC) and once with ice-cold 10mM Tris-HCl, pH 8. Chromatin complexes were eluted from the beads using elution buffer (10mM Tris-HCl pH 8, 0.1% SDS, 150mM NaCl, 5 mM DTT) for 1 hour at 65°C and treated with RNase A (Roche, Cat. No. 11119915001) for 30 min at 37oC. Proteinase K (Thermo Scientific, Cat. No. 25530049) treatment and reverse cross-linking was performed for 3 hours at 65°C. After crosslink reversal, immunoprecipitated DNA was extracted with AMP Pure XP beads (Beckman Coulter, Cat. No. A63881). ChIP DNA was quantified with Qubit dsDNA HS Assay kit (Thermo Scientific, Cat. No. Q32851). 1-2 ng ChIP DNA samples were used to prepare sequencing libraries, using the Ovation Ultralow System V2 kit (TECAN, Cat. No. 0344NB-A01).