GSM5283807: ChIP-seq TBI Input, 1-day rep 1; Rattus norvegicus; ChIP-Seq
Rats were placed in a chamber with continuous flow of 4% isoflurane. They were then removed from the chamber and rapidly decapitated. The brain was exposed and rolled out of the skull and placed on filter paper atop a glass dish sitting on ice. The cortex was hemisected rostral-to-caudal, then and rolled back (medial to lateral) exposing the underlying hippocampus. A curved, plastic spatula was used to roll the hippocampus out of the cortex and onto the filter paper. The same spatula was used to completely separate the hippocampus from the cortex and place it into a sterile microcentrifuge tube. (1) Total RNA was isolated from the samples using Ambion RNAqueous (Thermo Scientific cat # AM1912) and assayed using an Agilent BioAnalyzer instrument. We used TruSeq Stranded mRNA kits (Illumina P/N 20020594) to prepare the stranded mRNA libraries, that were sequenced on the Illumina HiSeq platform using a single-end 50-bp strategy. (2) Frozen hippocampus tissue samples were individually crosslinked with a 1% formaldehyde buffer solution for 10 min, then washed and isolated. The resuspended crosslinked pellets were treated with protease inhibitor and sheared by sonication. Samples were then washed off of unbound DNA and free proteins, and incubated with Histone H3K4me3 antibody (mAb, Active Motif cat # 61979) and magnetic beads (Dynabeads) for 2 hr at 4 ºC. After reaction, beads were magnetically separated, washed and de-crosslinked. The resulting DNA was then purified, size selected, and libraries were. prepared using Ovation Ultralow System V2 preparation kit (NuGEN P/N 0344). Input control libraries were prepared from DNA before antibody pulldown. The libraries were quantified and pooled prior to submission for sequencing.