GSM8655404: WA09_CTd120_K27ac_2; Homo sapiens; OTHER
Pluripotent stage cells (0 DIV) were harvested by incubation in Gentle Cell Dissociation Reagent (STEMCELL Technologies Cat. 07174) for 7 minutes at room temperature. GPC stage (180 DIV) cells were incubated in Accutase (Millipore Sigma Cat. A6964) diluted 50% in DPBS for 6 minutes at 37 degrees. After centrifugation, cells were resuspended in gentle nuclei isolation buffer (10mM HEPES pH 7.9, 10mM KCl, 0.5mM Spermidine, 0.10% IGEPAL, 1X cOmplete EDTA-free protease inhibitor cocktail (Roche Cat. COEDTAF-RO)). Following nuclei extraction, buffers were prepared and CUT&Tag was conducted (PSC: n=4 for each histone mark for WA09, n=2 H3K4me3 and H3K27ac, n=1 for H3K27me3 for C27; GPC: n=2 for WA09, n=3 for C27 for each histone mark) following the CUTANATM CUT&Tag protocol (EpiCypher, version 1.7)17. In brief, nuclei were adsorbed to Concanavalin A Conjugated Paramagnetic beads (EpiCypher Cat. 21-1401) and incubated with rabbit anti-H3K4me3 (Cell Signaling Technology Cat. 9751S), H3K27me3 (Cell Signaling Technology Cat. 9733S), or H3K27ac (Cell Signaling Technology Cat. 8173S) overnight at 4°C. The following day, anti-rabbit secondary antibody (EpiCypher Cat. 13-0047) was added, followed by pAG-Tn5 (EpiCypher Cat. 15-1117) to induce tagmentation of the chromatin. Tagmented DNA was indexed and amplified for sequencing, then sequenced on the NextSeq 550 System using the Mid-Output Kit (Illumina).