Efficient 3'-end tailing of RNA with modified adenosine for nanopore direct total RNA sequencing

Abstract

Direct sequencing of total cellular RNA enables a better understanding of a broad spectrum of RNA species controlling cellular processes and organismal function. Current nanopore direct RNA sequencing method, however, only captures polyadenylated RNA for sequencing. To address this issue, we developed a unique 3-end RNA tailing method to capture total RNA for nanopore direct RNA sequencing. Due to the distinct electrical signature of the added tail on nanopore, this method allows simultaneous detection of both non-polyadenylated and polyadenylated RNAs. We demonstrated the effectiveness of this method in capturing the dynamics of transcription and polyadenylation of chloroplast RNAs in plant cell. With its high efficiency in retaining total RNA on nanopore, this method has the potential to be broadly applied to RNA metabolism and functional genomics studies.