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

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 Overall design: 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).