title: GSM2461402: WT_IPBRG1; Homo sapiens; RIP-Seq
library_construction_protocol: For CLIP-seq 5 million cells were UV cross-linked using 2000 µJoules/cm2 followed by extraction  of nuclear proteins. UV crosslinked lysates from vector transfected, TGFR2G357W and ACTA2R179H  transfected cells were pre-supplemented with RNAse inhibitor (1:100), protease inhibitor cocktail,   phenylmethylsulfonyl fluoride (1mM, PMSF) and treated with turbo DNase I for 30min at 37C.  Lysates were then incubated with HDAC9 or BRG1 antibodies at 4C overnight. RNA extraction  from HDAC9 and BRG1 pull downs were performed using miRNeasy kit (Qiagen, USA) according  to the manufacturer’s instruction. RNA sequencing for human mRNA and lncRNAs were performed using Ribo-Zero™ Magnetic Kit and RnaseH is used to remove rRNA, remained RNA is then retrieved by ethonal precipitation. The retrieved RNA is fragmented using Ambion Fragmentation Solution. And according to Illumina solexa transcriptome sequencing protocol, random hexamer-primer was used to synthesize the first-strand cDNA. The second-strand cDNA is synthesized using buffer, dNTPs, RNase H and DNA polymerase I. Fragments are purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding A at 3’   end. After that, the fragments are ligated with sequencing adaptors. UNG is added before   synthesizing the second cDNA strand. After the second strand degraded, dUTP is then converted   to dTTP. Y-adaptor is added afterwards. Suitable fragments are selected as templates according   to the agarose gel electrophoresis results for PCR amplification. Generated libraries were sequenced using Illumina HiSeq™ technology.
attributes.tag: GEO Accession
attributes.value: GSM2461402