UV cross-linking RIP-seq of VSV-GFP-infected HEK293T cells overexpressed PTENα

To determine whether PTENα directly binds to viral RNA or host mRNA, we performed UV cross-linking RNA immunoprecipitation (RIP) sequencing of HEK293T cells transfected with vector encoding PTENα under VSV-GFP (green fluorescent protein-expressing vesicular stomatitis virus) infection Five 10cm2 dishes of HEK293T cells at 70% confluency were transfected with 10μg of FLAG-PTENα plasmid per dish. At 24 hours after transfection, cells were stimulated with VSV-GFP (MOI = 0.01). 14 hours later, cells were irradiated with 254nm UV wavelength of 400mJ/cm2 energy. Then, cells were collected and resuspended with co-IP lysis buffer under RNase Inhibitor treatment (1U/μl). After rotation at 4℃ for 15 min, the lysates were centrifuged at 13000 × g and the pellets were discarded. After saving 1/20 of the sample as input, the supernatant was incubated with ANTI-FLAG M2 beads and rotated at 4℃ for 2 hours to enrich the RNA-protein complex. Next, the beads were washed with IP washing buffer four times, and treated with DNase I (2U/μl) and RNase Inhibitor (1U/μl) at 22℃ for 15 min. Finally, the protein-RNA complexes were digested with Proteinase K (0.5mg/mL) and 0.1% SDS at 56℃ for 15 min and the bound RNA was separated using TRIzol reagent.