GSM8328998: IMR90, wild type, replicate 2, eclip; Homo sapiens; RIP-Seq
~20x10^6 IMR90 were UV-crosslinked (400 mJ/cm2 constant energy), lysed in iCLIP lysis buffer, and sonicated (on a BioRuptor Pico). Lysates were treated with RNase I (Ambion, AM2294) to fragment RNA, after which CTCF protein-RNA complexes were immunoprecipitated using the relevant antibody (anti-CTCF, Active Motif 61331). In parallel to the IP, an “input” library was generated for each replicate for which no antibody was used. Stringent washes were next performed, during which RNA was dephosphorylated with FastAP enzyme (Fermentas) and T4 PNK (NEB, M0201S). Subsequently, a 3' RNA adaptor oligonucleotide was ligated onto the RNA using a T4 RNA ligase (NEB, M0242S). Protein-RNA complexes were separated on an SDS-polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes, and RNA was isolated off the membrane. After precipitation, RNA was reverse-transcribed with AffinityScript reverse transcriptase (Agilent, 600107), free oligos were removed using ExoSap-IT (Thermo Fisher Scientific, 78201.1.ML), and a 3' DNA adaptor was ligated onto the cDNA product. Libraries were then amplified with 2x Q5 PCR mix (NEB)