title: GSM4830587: eCLIP_K562_rep1; Homo sapiens; RIP-Seq
library_construction_protocol: K562 and Hela cells (2e7 for each replicate) were UV crosslinked (254 nm, 400 mJ/cm2), then lysed and sonicated (Bioruptor) in eCLIP lysis buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 1% NP-40 (Igepal CA630), 0.1% SDS, 0.5% sodium deoxycholate, 1:200 Protease Inhibitor Cocktail I, in RNase/DNase-free H2O). RNA fragments were created by incubating lysates with RNase I (Ambion) and LUC7L2:RNA complexes were immunoprecipitated for 2h at 4C using Dynabeads bound to 4ug of LUC7L2-specific affinity-purified antibody (HPA051631). In parallel, libraries were generated from size-matched input (SMInput) samples containing RNAs present in the whole cell lysates, i.e. sans RBP-specific IP. For the IPs, a series of stringent washes (High salt wash buffer: 50 mM Tris–HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate (protect from light), in RNase/DNase-free H2O. ; Wash buffer: 20 mM Tris–HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20, in RNase/DNase-free H2O.) was followed by RNA dephosphorylation with FastAP (ThermoFisher Scientific) and T4 PNK (NEB) then ligation of an adaptor to the 3 ends of the RNAs with T4 RNA ligase 1 (NEB). Protein:RNA complexes were separated on 4-12%  polyacrylamide gels, transferred to a nitrocellulose membranes, and RNA was extracted from the membranes using Proteinase K (NEB). SMInput RNA were dephosphorylated and 3′-ligated and all samples were reverse transcribed with Superscript III (Invitrogen). Free primers were removed with ExoSap-IT (Affymetrix) and a DNA adaptor was ligated to the 3′ ends of the cDNA with T4 RNA ligase 1. cDNA was quantified by qPCR and PCR amplified using Q5 Master Mix (NEB) and resulting libraries were purified prior to Illumina sequencing.