GSM7966576: IP_8WG16_dhp1_ON_rep2; Schizosaccharomyces pombe; ChIP-Seq
Cells were fixed with 1% (ChIP) or 3% (Hi-C) formaldehyde 5mn at 32°C, 20mn at 19°C and quenched 5mn with 125mM glycine before being put on ice, washed with 1X PBS and frozen in liquid nitrogen. For ChIP, fixed cerevisae cells with Psm3-GFP tagged cohesin were added and cells were lysed with precellys, sonicated with S220 covaris, clarified twice by centrifugation and subjected to overnight immunoprecipitation before washes. DNA was eluted and purified with Qiagen Quiaquick columns. For Hi-C, cells were lysed with precellys, digested with DpnII overnight, filled in with dTTP, dGTP, dCTP and biotin-14-dATP for 45mn at 37°C, ligated for 4hr at 25°C and crosslinks were reversed overnight, and DNA was recovered with Phenol/Chloroform/IAA extraction and Amicon 30K column washes. For ChIPseq, NEBNext UltraII DNA Library Prep Kit E7645 and adapters E6609 were used. For Hi-C, libraries were performed with separate reagents, removing biotin from unligated ends with Klenow DNA Polymerase I, sonicating with Covaris, repairing extremities, A-tailing libraries and ligated NextFlex Adapters (CAT NOVA 514102) Libraries were sequenced with pairedend sequencing 150bp on a Novaseq6000