title: GSM3457401: H3K9ac.Control.EtOH.R1; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seq was performed as previously described (Nativio et al, Nat Neuroscience 2018) with modifications for mouse brain preparation. Briefly, ~20 mg dorsal hippocampus from each mouse was minced on ice and crosslinked with 1% formaldehyde for 10 min and quenched with 125 mM glycine for 5 min. Nuclei were prepared by dounce homogenization of crosslinked tissue in nuclei isolation buffer (50 mM Tris-HCl at pH 7.5, 25 mM KCl, 5 mM MgCl2, 0.25 M sucrose) with freshly added protease inhibitors and sodium butyrate. Nuclei were lysed in nuclei lysis buffer (10 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine) with freshly added protease inhibitors and sodium butyrate and chromatin was sheared using a Covaris S220 sonicator to ~250 bp size. Equal aliquots of sonicated chromatin were used per immunoprecipitation reaction with 5 ul H3K9ac antibody (Active Motif, cat # 39137; lot # 09811002 ) or 4 ul H3K27ac antibody (Abcam, cat #4729; lot # GR323132-1 (Piazza et al, Nat Commun 2018)) preconjugated to Protein G Dynabeads (Life Technologies). 10% of the chromatin was saved as Input. ChIP reactions were incubated overnight at 4°C with rotation and washed three times in wash buffer. Immunoprecipitated DNA was eluted from the beads, reversed crosslinked and purified together with Input DNA. 10 ng DNA (either ChIP or Input) was used to construct sequencing libraries using the NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, NEB). Libraries were multiplexed using NEBNext Multiplex Oligos for Illumina (dual index primers) and single-ended sequenced (75 bp) on the NextSeq 500 platform (Illumina) in accordance with the manufacturer's protocol.