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

Abstract

RNA polyadenylation is crucial for RNA maturation, stability and function, with polyA 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, tail composition and tail length estimates at single-molecule resolution. Taking advantage of a template switching-based protocol, Nano3P-seq can sequence any RNA 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 flowcells, as well as compatible software for polyA tail length and content prediction, which we term PolyTailor. We demonstrate that PolyTailor provides accurate estimates of transcript abundances, tail lengths and content information, while capturing both coding and non-coding RNA biotypes, including mRNAs, snRNAs, and rRNAs. This method can be applied to any RNA sample of interest (e.g. poly(A)-selected, ribodepleted, total RNA), and can be completed in one day. The Nano3P-seq protocol can be performed by researchers with moderate 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 non-coding transcriptome in a comprehensive and reproducible manner. Key PapersBe[g]ik O, Diensthuber G, Liu H, Delgado-Tejedor A, Kontur C, Niazi AM, Valen E, Giraldez AJ, Beaudoin JD, Mattick JS, Novoa EM. Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing. Nature Methods 20, 75-85 (2023). https://doi.org/10.1038/s41592-022-01714-w Delgado-Tejedor A, Medina M, Begik O, Cozzuto L, Lopez J, Blanco B, Ponomarenko J, Novoa EM. Native RNA nanopore sequencing reveals antibiotic-induced loss of rRNA modifications in the A- and P-sites. NatComm 15, 10054 (2024). https://doi.org/10.1038/s41467-024-54368-x