title: GSM2187972: H2030-BrM cells with control shRNA, brain slice, biological replicate 2; Homo sapiens; RNA-Seq
library_construction_protocol: Tissue culture cells or brain slices were lysed by pipetting in the TRAP lysis buffer containing 10mM HEPES, pH7.3, 150mM KCl, 5mM MgCl2, 0.5mM DTT, 1% NP-40, 100ug/ml Cycloheximide supplemented with protease inhibitors and RNase inhibitors. Lysates were cleared by centrifuge and the supernatans were supplemented with 1,2-Diheptanoyl-sn-Glycero-3-Phosphocholine (DHPC) at 30mM final concentration. Samples are further centrifuged and incubated with anti-EGFP monoclonal antibody conjugated Dynabeads for immunoprecipitation (IP). IP products were washed and RNA were subsequently purified by RNA mini kit (Ambion) from the beads. RNA samples were subjected to RNA-Seq analysis. TRAP RNA samples were quantified by Ribogreen and quality assessed by Agilent BioAnalyzer 2000. 100ng RNA with integrity number (RIN) > 9.5 from each sample was used for library construction with TruSeq RNA Sample Prep Kit v2 (Illumina) according to manufacturer’s instructions.  On average 40-45 million raw reads were generated for each sample.