Dynabeads protein g beads

RNA immunoprecipitation (RIP) for proteins such as YTHDC1 was carried out according to Jeon et al (Jeon and Lee, 2011 (link)) with modifications. Briefly, 5 millions of cells were UV-crosslinked at 254 nm (400 mJ/cm²) on ice and collected by scraping. Cells were lysed with 1ml cold lysis buffer (0.5% NP-40, 0.5% sodium deoxycholate, 20U/ml Superase-In, 1x Protease Inhibitor in PBS) at 4 °C for 25 minutes with rotation. Lysate was sonicated (Qsonica 800R, 25%, 5 minutes, 10 sec on/20 sec off interval), followed by 10U/ml Turbo DNase treatment for 15 minutes at 37°C. After centrifugation at 20,000g for 10 minutes, the supernatant was incubated with 1 μg Rabbit IgG or 1μg anti-YTHDC1 antibody (Abcam, Ab122340) immobilized on 20 μl Dynabeads™ Protein G beads overnight at 4°C. The next day, the beads were washed three times with 1ml ice-cold wash buffer (1% NP-40, 150mM NaCl, 0.5% sodium deoxycholate, 20U/ml Superase-In, Protease inhibitor in PBS), and treated with 10U Turbo DNase for 30min at 37°C. After three more washes with wash buffer supplemented with 10 mM EDTA, beads were digested with 200 μl Proteinase K buffer (100 mM Tris-HCl, 50 mM NaCl, 10 mM EDTA, 0.5% SDS and 500 μg/ml Proteinase K) for 30 minutes at 65°C. RNA was recovered by TRIzol reagent and examined by RT-qPCR.