CLIP-seq of VSV-infected HEK293T cells overexpressed PTENa

To determine whether PTENa directly binds to viral RNA, we performed Cross-Linking and Immunoprecipitation (CLIP) sequencing of HEK293T cells transfected with vector encoding PTENa under VSV (vesicular stomatitis virus) infection Overall design: Ten 10cm2 dishes of HEK293T cells at 70% confluency were transfected with 10µg of FLAG-PTENa plasmid per dish. At 24 h after transfection, the cells were stimulated with VSV (MOI = 0.01) in the presence of 100µM 4-thiouridine. 14 h later, cells were irradiated with 365nm UV wavelength of 150mJ/cm2 energy. Then, cells were collected and resuspended with co-IP lysis buffer. 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 predigested with RNase T1 (1U/µl) at 22? for 15 min, and the RNA-protein complex were enriched using ANTI-FLAG M2 beads with slow rotation at 4? for 2 h. Next, the beads were washed with IP washing buffer three times, and treated with DNase I (2U/µl) and RNase T1 (100U/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.