GSM5727304: 293T cells expressing 3xHA_EVI1+18; Homo sapiens; ChIP-Seq
Chromatin fractions from HEK293T cells were prepared using the fanChIP method, as described previously (doi.org/10.1016/j.xpro.2021.100404). Cells were suspended in CSK buffer (100 mM NaCl, 10 mM PIPES [pH 6.8], 3 mM MgCl2, 1 mM EGTA, 0.3 M sucrose, 0.5% Triton X-100, 5 mM sodium butyrate, 0.5 mM DTT, and protease inhibitor cocktail) and centrifuged (400× g for 5 min, at 4°C) to remove the soluble fraction. The pellet was resuspended in MNase buffer (50 mM Tris-HCl [pH 7.5], 4 mM MgCl2, 1 mM CaCl2, 0.3 M sucrose, 5 mM sodium butyrate, 0.5 mM DTT, and protease inhibitor cocktail) and treated with MNase at 37°C for 3–6 min to obtain oligonucleosomes. MNase reaction was then stopped by adding EDTA (pH 8.0) to a final concentration of 20 mM. An equal amount of lysis buffer (250 mM NaCl, 20 mM sodium phosphate [pH 7.0], 30 mM sodium pyrophosphate, 5 mM EDTA, 10 mM NaF, 0.1% NP-40, 10% glycerol, 1 mM DTT, and EDTA-free protease inhibitor cocktail) was added to increase solubility. The chromatin fraction was cleared by centrifugation (15,000 rpm for 5 min, 4°C) and subjected to immunoprecipitation with anti-HA antibody (3F10, Roche) and Protein-G magnetic microbeads (Invitrogen). Immunoprecipitates were then washed five times with washing buffer (1:1 mixture of lysis buffer and MNase buffer with 20 mM EDTA) and eluted in elution buffer (1% SDS and 50 mM NaHCO3). The eluted DNA material was fragmented by DNA shearing system (M220 Covaris). TruSeq ChIP Sample Prep Kit (illumina)