Endoribonuclease activity in hepatitis C virus-infected Huh7.5.1 cells

We used 2', 3'-cyclic phosphate cDNA synthesis and Illumina sequencing to identify and endoribonuclease cleavage sites in host and viral RNAs during HCV infection of Huh7.5.1 cells To make cDNA libraries, we exploited the 2’, 3’-cyclic phosphate at the end of RNA fragments produced by RNase L and other metal-ion-independent endoribonucleases. Using Arabidopsis thaliana tRNA ligase, RNA fragments with 2’, 3’-cyclic phosphates were covalently attached to defined RNA linkers containing an 8-base long unique molecular identifier (UMI) sequence. Libraries prepared in this manner contain cDNA derived from RNA fragments with 2', 3'-cyclic phosphates. The UMI sequence allows for detailed quantitation. We applied these methods to total RNA from Huh7.5.1 cells infected by hepatitis C virus 2a (JFH1).