title: GSM5525347: CR023_wo_S15; Homo sapiens; OTHER
library_construction_protocol: On the day of harvest, the cells were washed once with PBS, detached using Accutase, washed and resuspended in PBS. Briefly, 2x105 cells were washed twice (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x Roche complete protease inhibitors) and attached to pre-activated ConA-coated magnetic beads (Bangs Laboratories). Bead-bound cells were resuspended in 50 µL buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 0.5 mM Spermidine, 1x Roche complete protease inhibitors, 0.02% w/v digitonin, 2 mM EDTA) containing primary antibody (H3K9me3 (Ab8898, Abcam 1:50); H3k4me3 (Cat #39159, Active Motif 1;50) or IgG control antibody  (Ab97047, Abcam 1:50) at 4 ºC over-night. Washed with digitonin buffer, pA-MNase was added and incubated with the cells at 4 ºC for 1 h. Bead-bound cells were washed, resuspended in 100 µL digitonin buffer, and chilled to 0-2 ºC. Genome cleavage was stimulated by addition of 2 mM CaCl2 at 0 ºC for 30 min. The reaction was quenched by addition of 100 µL 2x stop buffer. After 10 min incubation at 37 ºC to release genomic fragments, cells and beads were pelleted by centrifugation (16,000 g, 5 min, 4 ºC) and fragments from the supernatant purified.