Abstract
Turnover of messenger RNAs (mRNAs) is a highly regulated process and serves to control expression of RNA molecules and to eliminate aberrant transcripts. Profiling mRNA decay using short-read sequencing methods that target either the 5' or 3' ends of RNAs, overlooks valuable information about the other end, which could provide significant insights into biological aspects and mechanisms of RNA decay. Oxford Nanopore Technology (ONT) is rapidly emerging as a powerful platform for direct sequencing of native, single-RNA molecules. However, as currently designed, the existing ONT platform is unable to sequence the very 5' ends of RNAs and is limited to polyadenylated molecules. Here, we present a detailed step-by-step experimental protocol for True End-to-end RNA Sequencing (TERA-Seq), a new method that addresses ONT's limitations, allowing accurate representation and characterization of RNAs at the level of single molecules. TERA-Seq describes both poly- and non-polyadenylated RNA molecules and accurately identifies their native ends by ligating uniquely designed adapters to the 5' ends (5TERA), the 3' ends (TERA3), or both ends (5TERA3) that are sequenced along with the transcripts.