GSM9701550: MV-4-11, endogenous DNMT1 dTAG line with ectopic overexpression of DNMT1 WT, dTAGV-1 for 5 days, rep 2; Homo sapiens; ChIP-Seq
Up to 30M cells were dual crosslinked in 10mL of 2mM DSG (in PBS) for 30 mins at RT with gentle agitation, followed by addition of formaldehyde (1% v/v final) for a following 15 minutes. Crosslinker was quenched with 40mL of 0.316M Tris (pH 8.0) and pelleted for 5 minutes at 400xg. Cells were washed twice in PBS and resuspended in ChIP lysis buffer (50mM Tris pH 8.0, 1% SDS, 10mM EDTA, supplemented with protease inhibitors). Chromatin was sonicated to approximately 250-1000bp using the ME220 Focused-ultrasonicator (Covaris) and 1mL Covaris milliTUBEs. Sonicated lysate was centrifuged at approx 20,000xg at 10°C for 15 minutes and supernatant was diluted in 1:10 wash buffer (55.5mM Tris pH8.0, 166mM NaCl, 1.1% Triton X-100, 2.2mM EDTA), containing protease inhibitors. Chromatin amounts were normalised prior to immunoprecipitation via Qubit Broad Range dsDNA assay. Immunoprecipitations were performed overnight at 4oC with rotation mixing. Antibody bound complexes were immobilised with 30μL of pre-washed Protein A Dynabeads (Thermofisher) for 4 hours at 4oC with rotation mixing and collected using a magnetic rack. Beads were washed five times with wash buffer (50mM Tris pH 8.0, 150mM NaCl, 1% v/v Triton X-100, 0.1% v/v SDS, 2mM EDTA) followed by a final wash with high salt buffer (50mM Tris pH 8.0, 500mM NaCl, 1% v/v Triton X-100, 0.1% v/v SDS, 2mM EDTA). Reverse crosslinking was achieved by incubating beads in elution buffer (20mM Tris pH 7.5, 200mM NaCl, 5mM EDTA, 1% v/v SDS, 200μg/mL Proteinase K (Roche) and 200µg/mL RNAse A (Qiagen)) at 65oC overnight with gentle agitation (1000 rpm). Immunoprecipitated DNA was purified using Qiagen MinElute columns (Qiagen) as per manufacturer's instructions. Sequencing libraries were prepared using the ThruPLEX® DNA-seq Kit (Takara Bio) as per manufacturer's instructions.