HyperTRIBE assay of VSV-GFP-infected HEK293T cells overexpressing ADAR1-CD, PTENa-N(WT)-ADAR1-CD, or PTENa-N(6R/D)-ADAR1-CD fusion protein

To explore the binding sites of PTENa on viral or host RNAs, we performed HyperTRIBE assay of HEK293T cells overexpressing ADAR1-CD, PTENa-N(WT)-ADAR1-CD, or PTENa-N(6R/D)-ADAR1-CD fusion protein under VSV-GFP (green fluorescent protein-expressing vesicular stomatitis virus) infection. Overall design: Five 10cm2 dishes of HEK293T cells at 70% confluency were transfected with 10µg of pCCL-EF1a-CMV-NGFR plasmid encoding ADAR1-CD, PTENa-N(WT)-ADAR1-CD, or PTENa-N(6R/D)-ADAR1-CD fusion protein per dish. At 24 hours after transfection, NGFR-positive cells were sorted and infected with VSV-GFP (MOI = 0.01). 6 hours later, cells were collected and total RNA was separated using TRIzol reagent.