GSM8308886: ChIRP_961C_LaCZ; Mus musculus; OTHER
The cells were cross-linked in PBS with 1% formaldehyde for 10min at room temperature with rotating and quenched by 125mM glycine for 5min. After washing cell pellet by ice-chilled PBS, the pellet was lysed in 1ml of lysis buffer (50 mM Tris-HCl pH 7.0, 10 mM EDTA, 1% SDS, add PMSF, PIC and Superase-in in fresh) per 100mg of cell pellet. Chromatin was fragmented by sonication with Bioruptor UCD200 (Diagenode) and cleared by centrifuge (16100G, 15min, 4C). Cleared chromatin was diluted in 2X volume of freshly-made hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris-HCl pH 7.0, 1 mM EDTA, 15% formamide, DTT, PMSF, P.I, and Superase-in) and hybridized with 100pmol of biotinylated DNA probes targeting HoxBlinc or LacZ at 37C overnight. DNA/RNA hybrids were precipitated using 100μL of Streptavidin-magnetic C1 beads (Invitrogen) and washed 5 times with washing buffer (2x SSC, 0.5% SDS). DNA was isolated using ZYMO ChIP DNA concentrator kit (D4014). ChIRP-seq library was prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB).