GSM7305489: Large preB cell, WT, CTCF HiChIP, rep1; Mus musculus; OTHER
Cells were crosslinked with 1% formaldehyde at RT for 10 minutes, and then quenched by the addition of 0.125 M glycine for 10 minutes. Cells were then washed with ice-cold PBS, snap frozen and stored at -80C before use. HiChIP assays were performed according to the previously published protocol (Mumbach et al., 2016) with minor modifications. Briefly, ~10 million cells were crosslinked with 1% Formaldehyde for 10 mins at room temperature and lysed with Hi-C lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP40) supplemented with protease inhibitors for 30 mins at 4C. Chromatin was digested by the MboI restriction enzyme for two hours at 37C. The 5' overhang of the digested DNA were filled with dCTP, dGTP, dTTP and biotinl14-dATP by DNA Polymerase I, Large Klenow Fragment, and then ligated by T4 DNA ligase for 4 hours at room temperature. Chromatin was sonicated in Nuclear Lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.6% SDS), 1:2 diluted in ChIP Dilution buffer (2% Triton X-100, 300 mM NaCl, 0.2% NaDoc), and then processed with regular chromatin immunoprecipitation using antibodies against H3K27ac, SMC1a, CTCF, or IKAROS. The DNA junctions labelled by Biotin-14-dATP were then enriched by Streptavidin C1 magnetic beads and processed with Nextera DNA library preparation (Illumina). The DNA libraries were size-selected by AMPure XP beads (300-700bp) and sequenced in house with an Illumina NEXTseq550 system and a Novaseq 6000 system at the Knapp Center for Biomedical Discovery, University of Chicago.