Protocol for generating native and unmodified bacterial RNA for nanopore direct RNA sequencing and downstream analysis.

Nanopore direct RNA sequencing (DRS) relies on adapter ligation to poly(A) tails, posing challenges for bacterial mRNAs, which naturally lack these structures. Here, we present a protocol for generating native and unmodified bacterial RNA for nanopore DRS and downstream analysis. We describe steps for preparing log-phase E. coli live cells, isolating native bacterial RNA, and synthesizing in vitro-transcribed (IVT) bacterial RNA. We then detail procedures for nanosphere DRS and a downstream data analysis pipeline for RNA modification detection. For complete details on the use and execution of this protocol, please refer to Guo et al.1.