Protocol for generating native and unmodified bacterial RNA for nanopore direct RNA sequencing and downstream analysis
Year: 2025
Type: article
Abstract: 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 • Protocol for nanopore direct RNA sequencing of bacterial RNA • Steps for bacterial RNA extraction and in vitro synthesis of unmodified RNA • Details on optimization of the protocol, limitations, and possible solutions • Guidance on data analysis of bacterial RNA modification detection Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Source: STAR Protocols
Authors: Zhihao Guo, Yanwen Shao, Lu Tan, Runsheng Li
Institution: City University of Hong Kong
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
OpenAlex ID: W4417535580