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

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.