GSM3391715: INPUT_ChIPSeq_dKO_rep2; Mus musculus; ChIP-Seq
Each E12.5 mouse brain was dissociated in 1 ml of cold PBS by pipetting. Protein−DNA complexes were crosslinked by incubating in 1% formaldehyde/PBS (Sigma F1635) for 10 min at RT with gentle rotation. Crosslinking was stopped with 125 mM glycine/PBS for 5 min and washed 3 times with cold PBS. Cells were lysed in 1 ml lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) supplemented with protease and RNAse inhibitors for 10 min at 4ºC with gentle rotation. Nuclei were pelleted at 1800 rcf for 2 min, washed with 1 ml wash buffer (10 mM Tris-HCl [pH 8.0], 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) supplemented with protease and RNAse inhibitors for 10 min, and lysed in 900 µl nuclear lysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-Lauroylsarcosine) for 10 min. Lysed nuclei (300 µl/1.5 ml sonication tube) were sonicated for 5 cycles of 15-sec on and 45-sec off in a Qsonica Q800R2 sonicator. Chromatin was quantitated and diluted with nuclear lysis buffer to ~500 ng/µl and further sonicated for 10 cycles of 15-sec on and 45-sec off to obtain an average DNA fragment size of ~300 bp. Sonicated chromatin was centrifuged at 14,000 rpm for 10 min and the supernatant chromatin was either used for ChIP or flash frozen and stored at −80ºC. Sequencing libraries were prepared from 10 ng of DNA using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina with NEBNext Q5 Hot Start HiFi PCR Master Mix according to the manufacturer's instructions (New England Biolabs) with the following modifications: a second 1:1 Ampure cleanup was added after adaptor ligation and the Ampure size selection step prior to PCR eliminated.
GEO Accession
GSM3391715