title: GSM1418792: RNA_shBRD4_YFP 1; Mus musculus; ncRNA-Seq
library_construction_protocol: RNA-seq: Cells were scraped in Swelling Buffer (10 mM TrisHCl, pH 7.5; 2 mM MgCl2; 3 mM CaCl2), and  lysed in Sucrose Lysis Buffer (5 mM TrisHCl, pH 7.5; 1 mM MgCl2; 3 mM CaCl2; 0.5% NP-40, 0.25 M sucrose). The nuclear pellet was resuspended in Chromatin Wash Buffer (10 mM TrisHCl, pH 7.5; 150 mM NaCl; 7.5 mM MgCl2; 0.1 mM EDTA; 0.5 M urea; 0.5% NP-40; 0.5 mM DTT). The nucleoplasmic supernatant was removed and the chromatin pellet was lysed with Trizol. Chromatin-associated RNA was extracted according to the Trizol protocol, treated with DNase I, and re-extracted with acid phenol chloroform and chloroform/isoamyl alcohol. RNA-seq: The chromatin-associated RNA was depleted of rRNA, and treated with RNA 5' Polyphosphatase and Terminator 5'-phosphate-dependent exonuclease. The 3' hydroxyl group of the nascent RNA was ligated with a pre-adenylated RNA 3' Adapter (TruSeq Small RNA Sample Preparation kit, Illumina) using T4 RNA ligase 2. The RNA was fragmented in 200 mM NaOH (most fragments <200 nt). The cap structure was removed with Tobacco Acid Pyrophosphatase. The 5'-ends of the hydrolyzed RNA fragments were phosphorylated with T4 Polynucleotide Kinase, 3' Phosphatase Minus, in the presence of ATP. Ligation of RNA 5'-adapter (TruSeq Small RNA Sample Preparation kit, Illumina), reverse transcription and PCR with multi-index primers were performed according to the Illumina protocol. Library fragments of ~320 bp (insert plus adaptor and PCR primer sequences) were band isolated. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on HiSeq 2000 following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM1418792