Nano3P-seq Nature Protocols

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 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. Barcode information: R9_Yeast: NB-01 TGIRT, NB-02 Induro R10_Yeast: NB-23 Induro, NB-24 Induro R10_DNAStd : NB-01 pA_standard_0, NB-02 pA_standard_15, NB-03 pA_standard_30, NB-04 pA_standard_60, NB-05 pA_standard_90, NB-06 pA_standard_120, NB-07 p29A_pU1_standard_30, NB-08 p27A_pU3_standard_30, NB-09 p25A_pU5_standard_30, NB-10 p25A_pC5_standard_30, NB-11 p25A_pG5_standard_30, NB-12 pA_internalG_standard_30

RNA多聚腺苷酸化（RNA polyadenylation）对于RNA的成熟、稳定性与功能至关重要，而polyA尾（polyA tail）的长度会显著影响信使RNA（mRNA）的翻译过程、翻译效率及降解速率。本研究提供了一套分步实验方案，用于开展纳米孔3'端捕获测序（Nanopore 3' end-capture sequencing，简称Nano3P-seq）——这是一种基于纳米孔的cDNA测序方法，可在单分子分辨率下同时捕获RNA丰度、尾段组成与尾长信息。该方案借助基于模板转换的实验流程，可从任意RNA分子的3'端进行测序，无需考虑其多聚腺苷酸化状态，且无需聚合酶链式反应（PCR）扩增或RNA接头连接。
本研究还提供了适配R10.4测序流通池（flowcells）的更新版Nano3P-seq实验方案，以及用于预测polyA尾长度与组成的配套软件PolyTailor。研究表明，PolyTailor可准确预测转录本丰度、尾长与组成信息，同时可覆盖编码RNA与非编码RNA两类生物型，包括信使RNA（mRNA）、小核RNA（snRNAs）以及核糖体RNA（rRNAs）。
本方案可降低Nano3P-seq的使用门槛，便于后续研究者便捷实施，以全面且可重复的方式探究编码与非编码转录组的尾段动态变化与异质性。
条形码信息：
R9_Yeast: NB-01 TGIRT, NB-02 Induro
R10_Yeast: NB-23 Induro, NB-24 Induro
R10_DNAStd: NB-01 pA_standard_0, NB-02 pA_standard_15, NB-03 pA_standard_30, NB-04 pA_standard_60, NB-05 pA_standard_90, NB-06 pA_standard_120, NB-07 p29A_pU1_standard_30, NB-08 p27A_pU3_standard_30, NB-09 p25A_pU5_standard_30, NB-10 p25A_pC5_standard_30, NB-11 p25A_pG5_standard_30, NB-12 pA_internalG_standard_30