title: GSM9020622: RAW 264.7, 4h, RRP1,CLIP,replicate5; Mus musculus; OTHER
library_name: GSM9020622
library_construction_protocol: Ultraviolet-crosslinked (400 mJ cm−2 ) RAW 264.7 were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 100mM NaCl, 1% NP-40, 0.1%SDS, 0.5% sodium deoxycholate and protease inhibitors) and sonicated. Lysates were treated with RNase I (Thermo Fisher Scientific) to fragment RNA and DNase I to remove DNA. Anti-FLAG® M2 Beads were washed three times using lysis buffer and incubated with proteins lyastes for 6h at 4°C. A 3' RNA adaptor was ligated onto the RNA with T4 RNA ligase (NEB). Protein-RNA complexes were run on a 4-12% gradient Bis-Tris Gels (Invitrogen), transferred to NC membranes, and RNA was isolated off the membrane identically to standard seCLIP. A fraction of sample was used for western blot of CLIPed Flag-tagged RRP1, indicating regions excised for seCLIP library preparation. After purification, RNAs were reverse transcribed with Superscript III reverse transcriptase (Invitrogen) with nested specific primer and a protected reverse PCR primer as previously reported, free primer was removed (ExoSap-IT, Affymetrix) and a 3' cDNA adaptor was ligated onto the cDNA product with T4 RNA ligase (NEB). Libraries were then amplified with Premix Taq PCR mix (Takara). Adapters and primers were designed according to the commercial indexing and sequencing primers.