Begik Oguzhan, Pryszcz Leszek P, Niazi Adnan Muhammad, Valen Eivind, Novoa Eva Maria
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.
Computational Biology Unit, Department of Informatics, University of Bergen, Bergen, Norway.
Nat Protoc. 2025 Jul 8. doi: 10.1038/s41596-025-01205-0.
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.
RNA多聚腺苷酸化对于RNA的成熟、稳定性和功能至关重要，多聚（A）尾的长度会显著影响mRNA的翻译、效率和降解。在此，我们提供了一份详细的步骤方案，用于进行纳米孔3'端捕获测序（Nano3P-seq），这是一种基于纳米孔的cDNA测序方法，能够在单分子分辨率下同时捕获RNA丰度、尾结构以及尾长度估计值。利用基于模板转换的方案，Nano3P-seq可以从任何RNA衍生分子的3'端进行测序，无论其多聚腺苷酸化状态如何，无需进行PCR扩增或RNA接头连接。我们提供了一种与R10.4流动槽兼容的更新版Nano3P-seq方案，以及用于多聚（A）尾长度和含量预测的兼容软件，我们将其称为“PolyTailor”。我们证明，PolyTailor能够准确估计转录本丰度、尾长度和组成，同时捕获编码和非编码RNA生物型，包括mRNA、小核仁RNA和核糖体RNA。Nano3P-seq可应用于通过不同方法制备的RNA样本（例如，poly(A)选择、核糖体去除或总RNA），并且可以在1天内完成。该方案需要分子生物学技术和纳米孔测序文库制备方面的经验，以及Linux Bash语法和R编程的基础知识。该方案使未来旨在以全面且可重复的方式研究编码和非编码转录组的尾动态和异质性的用户能够轻松使用Nano3P-seq并易于实施。
