title: GSM3145081: K562 par DMSO rep1; Homo sapiens; RNA-Seq
library_construction_protocol: eCLIP studies were performed in duplicates by Eclipse Bioinnovations Inc (San Diego, www.eclipsebio.com) according to the published single-end seCLIP protocol [[can cite pubmed ID 28766298]] with the following modifications. 10 million Molm13 cells were UV crosslinked at 400 mJoules/cm2 with 254 nm radiation, and snap frozen. Cells were then lysed and treated with RNase I to fragment RNA as previously described. RBM39 antibody (A300-291A lot 001, Bethyl) was then pre-coupled to Protein G Dynabeads (Thermo Fisher), added to lysate, and incubated overnight at 4 deg C. Prior to immunoprecipitation, 2% of the sample was taken as the paired input sample, with the remainder magnetically separated and washed with lysis buffer only (as the standard high-salt eCLIP wash buffer gave poor immunoprecipitation yield). RNA adapter ligation, IP-western, reverse transcription, DNA adapter ligation, and PCR amplification were performed as previously described. eCLIP library preparation  eCLIP studies were performed in duplicates by Eclipse Bioinnovations Inc (San Diego, www.eclipsebio.com) according to the published single-end seCLIP protocol [[can cite pubmed ID 28766298]] with the following modifications. 10 million Molm13 cells were UV crosslinked at 400 mJoules/cm2 with 254 nm radiation, and snap frozen. Cells were then lysed and treated with RNase I to fragment RNA as previously described. RBM39 antibody (A300-291A lot 001, Bethyl) was then pre-coupled to Protein G Dynabeads (Thermo Fisher), added to lysate, and incubated overnight at 4 deg C. Prior to immunoprecipitation, 2% of the sample was taken as the paired input sample, with the remainder magnetically separated and washed with lysis buffer only (as the standard high-salt eCLIP wash buffer gave poor immunoprecipitation yield). RNA adapter ligation, IP-western, reverse transcription, DNA adapter ligation, and PCR amplification were performed as previously described. RNA-seq library preparation  CRISPR-mediated RBM39 knockout cells were harvested day 6 post-transduction and wash with 1x PBS. To extract total RNA, we used RNeasy Plus Mini Kit (Qiagen) and QIAshredder (Qiagen), which were performed according to manufacturer's protocol. We isolated poly(A) mRNA using magnetic isolation method (NEB) using ~1 ug of total mRNA. To generate RNA-sequencing libraries, we used NEXTflex Rapid Directional (Bioo Scientific) protocol and NEXTflex RNA-seq barcodes (Bioo Scientific), which were carried out according to manufacturer's protocol. RNA-seq barcoded libraries were then sequenced using Hi-Seq 4000 (150 cycles paired end).
attributes.tag: GEO Accession
attributes.value: GSM3145081