title: GSM8827987: MEF_PUS10KO_H3K4me3; Mus musculus; OTHER
library_name: GSM8827987
library_construction_protocol: 5 × 105 WT and Pus10KO cells were washed twice (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× Roche cOmplete protease inhibitors) and attached to 10 μL (per cell line per antibody) ConA-coated magnetic beads (Bangs Laboratories) that had been pre-activated in binding buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Cells were then resuspended in 50 μL buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 0.5 mM Spermidine, 1× Roche complete protease inhibitors, 0.05% w/v digitonin, 2 mM EDTA) containing 1:50 dilution of the primary antibody (goat anti-rabbit IgG (Abcam, CAT#ab97047), rabbit anti-H3K4me3 (Active motif CAT#39159) and incubated at 4 °C overnight. Beads were washed thoroughly with digitonin buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1× Roche cOmplete protease inhibitors, 0.05% digitonin). After the last wash, pA-MNase (a generous gift from Steve Henikoff) was added in digitonin buffer and incubated with the cells at 4 °C for 1 hr. Bead-bound cells were washed twice, resuspended in 100 μL digitonin buffer, and chilled to 0–2 °C. Genome cleavage was stimulated by addition of 2 mM CaCl2 at 0 °C for 30 min. The reaction was quenched in 100 mL 2 stop buffer (0.35 M NaCl, 20 mM EDTA, 4mM EGTA, 0.05% digitonin, 50 ng/mL glycogen, 50 ng/mL RNase A and vortexing. After 10 min incubation at 37 °C to release cleaved fragments, cells and beads were pelleted by centrifugation (16,000 × g, 5 min, 4 °C) and the supernatant purified by a PCR clean-up kit (Macherey-Nagel). Illumina sequencing libraries were prepared using the Hyperprep kit (KAPA) with unique dual indexed adapters (KAPA), pooled and sequenced on a Nextseq500 instrument (Illumina).