ネイティブかつ未修飾の細菌RNAをナノポア直接RNAシーケンシングおよび下流解析用に生成するためのプロトコル

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.