GSM6820662: HeLa_WT_biol rep3; Homo sapiens; RIP-Seq
CLIP was performed as previously described with slight modifications (Liu et al., 2016). HeLa cells were seeded in 10 cm dish and then transfected with plasmids expressing V5-tagged GlyRSWT, GlyRSP234KY or GlyRSP234KY-L42A. After 24 hours, cells were treated with 250 μM AS for 1 hour, then the medium was aspirated and the cells were washed twice with 5 mL ice-cold PBS. Subsequently, 4 ml of ice-cold PBS was added to the culture dish and the cells were irradiated once with 400 mJ/cm2 at 254 nm in UV-linker (UVP-CL-1000) on ice. After irradiation, the cells were scraped, collected, and lysed on ice with 1 mL lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1×Protease Inhibitor Cocktail and 2.5 U/μL RNase inhibitor). After 30 min of incubation, the cell lysates were centrifuged at 12, 000 rpm, at 4℃ for 15 min, and then the supernatant was carefully collected and added 6 μg G3BP1 antibody (Santa cruz), then the mixture was incubated at 4˚C with gentle rotation overnight. The next day, the mixture was subsequently added 80 μL of protein A/G beads (Invitrogen), and continued to incubate at 4˚C with gentle rotation. After 4 hours, the supernatant was discarded and the beads were washed 3 times with 1 mL of high-salt buffer (5 × PBS supplemented with 0.1% SDS and 1% Nonidet P-40) and another 3 times with 1 mL of wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2 and 0.2% Tween-20). Subsequently, the beads were treated with 2 mg/mL proteinase K at 50˚C for 1 h, then magnetically separated, discarded, and the supernatant was added 1 mL Trizol reagent. Finally, protein-bind RNA were extracted using Trizol reagent and were precipitated by ethanol. The CLIP RNA was assessed by Agilent 2200 TapeStation(Agilent Technologies, USA) and Qubit(Thermo Fisher Scientifc,USA). Briefly, CLIP RNA were fragmented to approximately 200bp. Then, the RNA fragments were subjected to first strand and second strand cDNA synthesis following by adaptor ligation and enrichment with a low-cycle according to instructions of NEBNext® Ultra RNA LibraryPrep Kit for Illumina (NEB, USA). The final library product was assessed with Agilent 2200 TapeStation andQubit®(Life Technologies, USA) and then sequenced on Illumina （Illumina,USA）platformat with pair-end 150bp at Ribobio Co. Ltd (Ribobio, China).