title: GSM5050901: ChIP-seq_E14_Dnmt3a_cKO_H3K27me3_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: The frontal cortex tissue was rapidly dissected from 1-2 adult mice on ice, and the INTACT method was used to affinity purify GFP+ nuclei. Sorted nuclei were crosslinked for 15min in 1% formaldehyde solution and quenched afterward with glycine at a final concentration of 0.125 M. After crosslinking, nuclei were sonicated in Lysis buffer (50 mM Tris HCl pH 8, 20 mM EDTA, 1% SDS, 1X EDTA free protease inhibitor cocktail). ChIP assays were conducted with antibodies against H3K27me3 (39156, Active Motif), H3K27ac (39133, Active Motif) and H3K4me3 (04-745, Millipore Sigma). Mouse IgG (015-000-003, Jackson ImmunoResearch) served as negative control. H3K4me3 ChIP-seq assays were conducted with 100K nuclei and 500K nuclei were used for H3K27me3 and H3K27ac ChIP-seq assays. The respective antibodies and IgG were coupled for 4-6 hours to Protein G Dynabeads (50µl, 10004D, Thermo Fisher Scientific). Equal amounts of sonicated chromatin were diluted with 9 volumes of Binding buffer (1% Triton X-100, 0.1% Sodium Deoxycholate, 2X EDTA free protease inhibitor cocktail) and subsequently incubated overnight with the respective antibody-coupled Protein G beads. Beads were washed successively with high salt buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100) low salt (50 mM Tris HCl pH 7.4, 500 mM NaCl, 2 mM EDTA, 0.5% Triton X-100) and wash buffer (50 mM Tris HCl pH 7.4, 50 mM NaCl, 2 mM EDTA) before de-crosslinking, proteinase K digestion and DNA precipitation. Libraries were generated with the Accel-NGS 2S Plus DNA Library Kit (21024, Swift Biosciences) .