title: GSM2756421: eCLIP_MCF7_cytoplasm_input; 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. Cellular fractionation was performed as previously described (Wysocka et al. 2001). Each fraction was raised to 1 mL final volume using Buffer D (20 mM HEPES pH 7.5, 210 mM NaCl, 7.5% glycerol, 0.75 mM MgCl2, 0.25 mM PMSF, and 1x proteinase inhibitor) then incubated with TURBO DNase (40U for chromatin sample, 10U for soluble samples) for 30 minutes at 37ºC, then quenched with 10 mM EDTA. Fractions were then further digested with Bioruptor and RNase (but no additional DNase) as specified in the original eCLIP protocol, then immunoprecipitated overnight with antibody to hnRNP B1(IBL 18941, 2.5 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: GSM2756421