GSM9630669: HNRNPC_inRBM25_MUT_2; Homo sapiens; RNA-Seq
Cell pellets were lysed with PBS lysis buffer (1x PBS, 0.1% SDS, 0.5% NaDoc, 0.5% NP-40, 1 protease inhibitor tablet (Roche)) for 10 minutes on ice. Lysate was then treated with RQ1 DNase (Promega) for 5 minutes at 37°C with shaking at 1100RPM. The lysates were then briefly treated with RNase I at 1:100 or 1:1000 dilutions at the same condition as DNase treatment. Lysates were clarified by centrifuging on tabletop 4°C centrifuge for 10 minutes. For V5-tagged protein samples, V5 antibody (Addgene, cat. #218107-rAB) was used. For endogenous HNRNPC IP in competition CLIP samples, anti-HNRNPC antibody (Invitrogen, cat. #PA5-22280) was used. Immunoprecipitated samples were labeled with radioactivity (gamma-32P-ATP (Revvity)) and ran on SDS-PAGE in non-reducing conditions. The gels were transferred to a nitrocellulose membrane, and bands 10-30kDa above hnRNPC (32kDa) were excised. The excised membranes were treated with proteinase K (Roche) to release the protein-bound RNAs from the membrane. CLIP footprints were reverse-transcribed using the BrdU incorporation and bead-capture strategy described previously (PMID: 24613350).