Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing

Koichi Ogami; Yuka Oishi; Shin-Ichi Hoshino

doi:10.1016/j.xpro.2023.102340

Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing

STAR Protoc. 2023 May 26;4(2):102340. doi: 10.1016/j.xpro.2023.102340. Online ahead of print.

Authors

Koichi Ogami¹, Yuka Oishi², Shin-Ichi Hoshino³

Affiliations

¹ Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 Japan; Division of Molecular Oncology, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
² Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 Japan.
³ Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 Japan. Electronic address: hoshino@phar.nagoya-cu.ac.jp.

Abstract

Poly(A) tail metabolism contributes to post-transcriptional regulation of gene expression. Here, we present a protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing, which excludes truncated RNAs from the measurement. We describe steps for preparing recombinant eIF4E mutant protein, purifying m7G- capped RNAs, library preparation, and sequencing. Resulting data can be used not only for expression profiling and poly(A) tail length estimation but also for detecting alternative splicing and polyadenylation events and RNA base modification. For complete details on the use and execution of this protocol, please refer to Ogami et al. (2022).¹.

Keywords: Cell Biology; Molecular Biology.