title: GSM2756414: eCLIP_MCF7_B1_B; Homo sapiens; RIP-Seq
library_construction_protocol: eCLIP-seq was performed as previously described (Van Nostrand et al. 2016) with minor modifications. Briefly, confluent cells were UV-crosslinked at 150 mJ and 254 nm wavelength in 13 mL of PBS in 15 cm plates; these cells were then spun down, flash frozen in liquid nitrogen, and stored at –80ºC. Nuclear isolation was performed as previously described (Kung et al. 2015), followed by further digestion by Bioruptor, RNase I, and Turbo DNase as specified in the original eCLIP protocol. Overnight immunoprecipitation was performed using 2 mg Protein G Dynabeads per 10 µg antibody, and appropriate antibodies for hnRNP A2/B1 (Abcam ab31645, 9 µg/IP), hnRNP B1 (IBL 18941, 2.5 µg/IP), or IgG (Novus NB810-56910, 5 µg/IP). Following end repair and 3′ adaptor ligation (using X1A and X1B adaptors as previously described), size selection was carried out using Nupage 4-12% Bis-Tris protein gels followed by overnight transfer at 30V to nitrocellulose membranes. Protein-RNA complexes on nitrocellulose were then digested with Proteinase K (Roche) and reverse transcribed using SuperScript IV (Thermo Fisher). These cDNAs were then prepared for sequencing as described using Illumina TruSeq HT dual indexed primers, and sequenced on an Illumina NextSeq 500 for high-output 2x75 bp run.
attributes.tag: GEO Accession
attributes.value: GSM2756414