GSM5291196: HSPC_donor2_H3K27ac; Homo sapiens; OTHER
1-5x105 cells were used for H3K27ac/H3K14ac/H3K27me3 antibodies, and 2 million cells were used for other antibodies. Digitonin was diluted at a final concentration of 0.1% (w/v) in the antibody buffer (150 mM NaCl, 20 mM HEPES-KOH, 0.5 mM Spermidine, 2 mM EDTA, protease inhibitor cocktail plus digitonin), and Dig-wash buffer (150 mM NaCl, 20 mM HEPES-KOH, 0.5 mM Spermidine, protease inhibitor cocktail plus digitonin). For primary patient samples from frozen vials and CD34+ HPSCs, live cells were purified with the Dead Cell Removal kit (MACS) before wash and a final concentration of 0.05% of Digitonin was used for subsequent steps. Cells were washed twice in Wash buffer (150 mM NaCl, 20 mM HEPES-KOH,0.5 mM Spermidine, 2 mM EDTA, protease inhibitor cocktail) and incubated with 20 µl of Concanavalin A-coated beads for 10 min with rotation at room temperature. Antibody buffer containing indicated antibodies was added to the cell-attached beads and incubation was at 4°C with rotation overnight. Beads were washed with Dig-wash buffer and incubated with 300 µl Dig-wash buffer containing ~700 ng/ml pA-MN at 4°C for 1 hr. Beads were washed twice with Dig-wash buffer and chilled on a heated block sitting on ice. Digestion was activated through adding CaCl2 and chromatin was digested for 60 min when applying ZMYND8 antibody (Bethyl), and 30 min for other antibodies. Equal volume of 2x Stop solution (340 mM NaCl, 4 mM EGTA, 20 mM EDTA, 50 µg/ml RNase A, 0.02% Digitonin, 50 µg/ml Glycogen, 2 pg/ml heterogenous spike-in DNA) was added and beads were incubated at 37°C for 10 min to release the digested chromatin. Supernatant was collected and DNA was extracted through Phenol-Chloroform extraction. Library construction was prepared with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina with small modifications. For H3K27ac, H3K14ac and H3K27me3, adapter was diluted on a ratio of 1:25, and repaired DNA was first selected with 25 µl Ampure Beads (Beckman) and second selected with 45 µl beads, followed by 9 cycles of PCR amplification. For chromatin regulators, library preparation was previously described (Liu et al., Cell., 2018). Adapter was diluted on a ratio of 1:50 and 10-12 cycles of PCR amplification was used.