H3K27 ChIP-Seq of mouse line WSR-1 prefrontal cortex following chronic ethanol vapor exposure and 21 days of withdrawal
Twenty-one days after cessation of ethanol vapor or control exposure, mice were euthanized and the prefrontal cortex was rapidly isolated, snap-frozen in liquid nitrogen, and stored at -80 C until processing. Each prefrontal cortex was homogenized in a 10 mL Dounce homogenizer in RPMI buffer, cross-linked with a final concentration of 1.6% formaldehyde, and fragmented by sonication using a Bioruptor-Pico (Diagenode; Denville, NJ) to achieve fragment sizes ranging from ~100-500 bp. Protein A/G PLUS-Agarose Beads were used to pre-clear the fragmented chromatin samples for 2h at 4 C with slow end-over-end rotation. Primary antibody (Millipore Anti-Trimethyl-Histone H3 (Lys27), Cat# 07-449) was added to supernatant after clearing along with fresh agarose beads and incubated overnight at 4 C with gentle end-over-end rotation. The following day, beads were washed with low salt buffer, high salt buffer, lithium chloride buffer, and TE buffer followed by elution from the beads and DNA isolation using the ChIP DNA Clean & Concentrator Kit (Zymo Research; Irvine, CA).  DNA was quantitated using the Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific; Waltham, MA) according to the manufacturers protocol. Sequencing libraries were prepared using the NEBNext ChIP-seq Library Prep Master Mix Set and Multiplex Oligos for Illumina Index Primer Set 1 according to the manufacturers protocol using 10 ng of ChIP DNA and size selection for 150 bp inserts (New England Biolabs; Ipswich, MA). Short read sequencing assays were performed by the OHSU Massively Parallel Sequencing Shared Resource.