Nano3P-seq: charting the coding and noncoding transcriptome at single-molecule resolution.

RNA polyadenylation is crucial for RNA maturation, stability and function, with poly(A) tail lengths significantly influencing mRNA translation, efficiency and decay. Here, we provide a step-by-step protocol to perform Nanopore 3' end-capture sequencing (Nano3P-seq), a nanopore-based cDNA sequencing method to simultaneously capture RNA abundances and tail-composition and tail-length estimates at single-molecule resolution. Taking advantage of a template switching-based protocol, Nano3P-seq can sequence any RNA-derived molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or RNA adapter ligation. We provide an updated Nano3P-seq protocol that is compatible with R10.4 flow cells, as well as compatible software for poly(A) tail length and content prediction, which we term 'PolyTailor'. We demonstrate that PolyTailor provides accurate estimates of transcript abundances and tail lengths and composition, while capturing both coding and noncoding RNA biotypes, including mRNAs, small nucleolar RNAs and ribosomal RNAs. Nano3P-seq can be applied to RNA samples prepared by using different methods (e.g., poly(A)-selected, ribodepleted or total RNA) and can be completed in 1 day. The protocol requires experience in molecular biology techniques and nanopore sequencing library preparation and basic knowledge of Linux Bash syntax and R programming. This protocol makes Nano3P-seq accessible and easy to implement by future users aiming to study the tail dynamics and heterogeneity of both coding and noncoding transcriptomes in a comprehensive and reproducible manner.