title: GSM1418806: RIP_shBRD4_YFP-BRD4; Mus musculus; RIP-Seq
library_construction_protocol: RIP-seq: Nuclei were isolated, and incubated at 30°C for 5 min in Nuclear Run-On Master Mix without Sarkosyl (5 mM TrisHCl pH8.0, 150 mM KCl, 2.5 mM MgCl2, 0.5 mM DTT, 0.25 mM ATP, 0.25 mM GTP, 0.0012 mM CTP, 0.25 mM Br-UTP). The run-on nuclei were digested with DNase I. Insoluble materials were removed by centrifugation, and the supernatant was pre-cleared with Dynabeads Protein A, and then  incubated with the Dynabeads Protein A coupled with anti-YFP antibody. The beads were washed five times with RIP Wash Buffer, and RNA was extracted with Trizol. RIP-seq: The precipitated 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 by base hydrolysis in 0.2 M NaOH, neutralized and immunoprecipitated with anti-BrdU agarose beads. The 5’ cap structure was removed with Tobacco Acid Pyrophosphatase. The 5'-ends of the hydrolyzed RNA fragments were phosphorylated with T4 Polynucleotide Kinase. Ligation of RNA 5'-adapter (Illumina), reverse transcription and PCR with multi-index primers were performed according to the Illumina protocol.
attributes.tag: GEO Accession
attributes.value: GSM1418806