GSM9271142: Day5_PECHiC_PRC2perturb_PROTAC_Batch2_ BioRep4; Homo sapiens; OTHER
Cells were dissociated into single-cell suspensions using TrypLE. 1 million cells per condition were fixed in 1X D-PBS (Sigma-Aldrich) containing 2 % paraformaldehyde (Electron Microscopy Sciences) for 10 min at room temperature. Crosslinking was quenched with incubation in 0.125 M glycine for 5 min at room temperature followed by 5 min on ice. Cells were pelleted by centrifugation, washed with PBS, and lysed for 30 min on ice in Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630, protease inhibitors). All centrifuging steps during the lysis, prior to DpnII digestion, were carried out in a swinging-bucket centrifuge at 4°C, 300 x g for 5 min.The released nuclei were permeabilised and digested overnight with DpnII (NEB). Restriction overhangs were filled in with biotin-dATP (Jena Bioscience), and proximity ligation was performed for 4 h at 16 °C using T4 DNA ligase (Invitrogen). Crosslinks were reversed overnight at 65 °C with proteinase K, and DNA was purified with AMPure XP beads (Beckman Coulter). DNA was fragmented by tagmentation to generate fragments ≤1,000 bp fragments, and biotinylated junctions were enriched using MyOne C1 streptavidin beads (Life Technologies). Libraries were amplified directly on beads with 5 PCR cycles, purified with AMPure XP beads, and subjected to poised enhancer capture using a custom-designed Agilent SureSelect system, following the manufacturer's protocol. Libraries were sequenced as 150 bp paired-end reads.