GSM3258516: IP-MTG1-3; Homo sapiens; RIP-Seq
Cells stably expressing Flag-tagged MTG1 proteins were grown in medium supplemented with 100 uM 4–thiouridine (SU) for 14 hr, and subjected to UV-mediated protein-RNA crosslinking once with 150 mL/cm2 at 365 nm using a Stratalinker UV crosslinker (Stratagene) before disrupting them with 1% NP40 lysis buffer (PBS, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with EDTA-free protease inhibitor cocktail (Roche), and ribonucleoside vanadyl complexes (NEB) for 10 min at 4˚C. 4SU-labeled non-irradiated cells were used as control. The lysate was spun at 30,000 rpm for 20 min at 4˚C in a TLA 100 rotor, and the eluted protein-RNA complexes were applied for immunoprecipitation with anti-FLAG M2 beads. Bound protein-RNA complexes were treated with DNase for 30 min at 37˚C, and RNA was extracted from the beads by proteinase K treatment and QIAzol extraction (Qiagen). KAPA Stranded RNA-Seq Library Preparation Kit