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 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).

本研究采用2',3'-环状磷酸cDNA合成法与Illumina测序技术，鉴定Huh7.5.1细胞感染丙型肝炎病毒（hepatitis C virus，HCV）过程中宿主RNA与病毒RNA的核糖核酸内切酶切割位点。
总体实验设计：构建cDNA文库时，我们利用了核糖核酸酶L（RNase L）及其他不依赖金属离子的核糖核酸内切酶所产生的RNA片段末端的2',3'-环状磷酸基团。使用拟南芥（Arabidopsis thaliana）tRNA连接酶，我们将带有2',3'-环状磷酸基团的RNA片段与带有8碱基长度的独特分子标识符（unique molecular identifier，UMI）序列的定制RNA接头进行共价连接。以此方式构建的文库包含源自带有2',3'-环状磷酸基团的RNA片段的cDNA；独特分子标识符可实现精准的定量分析。我们将上述方法应用于感染丙型肝炎病毒2a型（JFH1）的Huh7.5.1细胞的总RNA样本。