GSM8155939: input_EV1; Homo sapiens; ChIP-Seq
Adherent cells were directly fixed into the dishes with 2mM DSG (Santa Cruz, Cat# sc-285455) + 1mM MgCl2 in 8ml 1X PBS for 45min at room temperature. Then cells were cross-linked with 1% methanol-free formaldehyde (Pierce Life Technologies, Cat# 28906) and quenched with 0.125 M glycine. Cells were lysed with lysis buffer LB1 (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP40, 0.25% TritonX-100, Protease inhibitor cocktail (Sigma, Cat # P8340)) twice during 10 minutes at 4 °C. Nuclear lysis was performed using LB2 buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, Protease inhibitor cocktail (Sigma, Cat # P8340)) for 10 minutes at 4°C. The cell pellet was rinsed twice with SDS shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.15% SDS, Protease inhibitor cocktail), resuspended in 1ml SDS shearing buffer and transferred to a milliTUBE 1 ml AFA fiber. Chromatin was sonicated on a E220 focused ultrasonicator (Covaris) using the following settings: 18 min, 200 cycles, 5% duty, 140W. The IP reactions were performed following the protocols of the Diagenode iDeal ChIP-seq kit for Transcription Factors (Diagenode, Cat# C01010170). For each IP reaction, 4 million cells were used. Sheared chromatin was immunoprecipitated overnight at 4°C with HA (Abcam Cat #ab9110, 2µg), RBPJ (CST Cat #5313, 1:50) or SOX2 (CST Cat #23064, 1:50) antibodies. Eluted DNA was quantified with Qubit (Thermo Fisher) and Agilent Fragment Analyzer (Advanced Analytical). We performed a "NEBNext Ultra II DNA" library prep starting from 3 ng of DNA for 'Chip' samples and 10ng of DNA for 'input' samples (see table below), according to New England Biolabs' protocol NEBNext Ultra™ II DNA Library Prep Kit, Version 6.1_5/20 (internal exp code RPX030). We quantified the libraries by qubit DNA HS and performed profile analysis by TapeStation TS4200.