GSM4831202: 12-6A-Old-Input; Homo sapiens; ChIP-Seq
Briefly, 200 mg brain tissue from each patient was minced on ice and nuclei were prepared by dounce homogenization 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, followed by ultracentrifugation on a 1.8 M sucrose cushion. Nuclei pellet was resuspended in 2 ml PBS and crosslinked in 1% formaldehyde for 10 min at room temperature. Crosslinking reactions were quenched with addition of glycine to 125 mM for 5 min followed by two washes in cold PBS. 2 x 106 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 H4K16ac antibody (Millipore, #07-329) preconjugated to Protein G Dynabeads (Life Technologies), and 10% of the amount 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 washed beads, purified and used to construct sequencing libraries with 5 ng of DNA (ChIP or Input) using the NEBNext Ultra 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.