GSM2790409: CTCF ZT22; Mus musculus; ChIP-Seq
Genomic DNA was isolated by phenol:chloroform extraction followed by cloroform wash. Nuclei isolation: Mouse liver tissue (100mg) was dounced with 15 mL of cold swelling buffer (10mM HEPES, 2mM MgCl2, 3mM CaCl3) 10 times with piston A. After 20 min incubation on ice, the homogenate was dounced again 20 times with piston B, after which additional 15mL of cold swelling buffer was added. The homogenate was filtered through a 100mm cell strainer and spun at 400g in 4°C for 10 min. The supernatant was discarded, and the pellet resuspended in 10mL of cold swelling buffer containing 10% glycerol. 10mL of cold lysis buffer (swelling buffer + 10% glycerol +1% Igepal) was slowly added with occasional vortexing. After 5 min incubation on ice, 30mL of cold lysis buffer was added, after which it was spun down at 600g in 4°C for 5 min. The supernatant was discarded and the pellet resuspended with 25mL of cold lysis buffer and spun down at 600g at 4°C for 5 min again. The supernatant was discarded, and the pellet contained isolated, undisrupted nuclei. For ChIP and Hi-C experiments, these pure nuclei were crosslinked in 10 mL of PBS with 1% formaldehyde for 20 min at room temperature and quenched with 1/20 volume of 2.5M glycine solution for 5 min. The crosslinked nuclei were spun down at 600g at 4°C for 5 min, after which they were resuspended in Hi-C lysis buffer (10mM Tris-HCl pH8, 10mM NaCl, 0.2% Igepal) and spun down at 600g in 4°C for 5 min again. The supernatant was discarded, and crosslinked nuclei was used for ChIP and Hi-C. ChIP-seq: 1-5 million crosslinked nuclei were used per immunoprecipitation. Nuclear extract were prepared by sonication in lysis buffer (50mM Tris-HCl pH 8, 0.1% SDS, 10mM EDTA) using the Bioruptor (Diagenode) for a fragment range of 200-1000bp. Chromatin was immunoprecipitated in ChIP buffer (50mM Tris-HCl pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% NaDOC) using 5-10ug of antibody, and reverse crosslinked overnight at 65°C in SDS buffer (50mM Tris-HCl, 10mM EDTA, 1% SDS, pH 8). DNA was isolated using phenol/chloroform/isoamyl alcohol and second chloroform wash. Hi-C: In situ Hi-C was performed with mouse liver tissues using MboI restriction enzyme based on the protocol provided by Rao et al. Cell 2014 (PMID: 25497547) with minor changes described below. In addition, biotin from unligated ends were removed by incubating 5ug of DNA in 50ul T4 DNA polymerase reaction (0.1ug/ul BSA, 1xNEB buffer 2, 25uM dGTP, 15U T4 DNA polymerase). The reaction was carried out at room temperature for 4 hours and stopped by the addition of 2ul of 0.5M EDTA pH 8.0 per 50ul reaction. Isolated Hi-C DNA was amplified by fewer than 8 PCR cycles to reduce PCR duplicates.