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

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• RNA polyadenylation is vital for RNA processing, affecting stability, translation, and decay.
• Poly(A) tail length is a key determinant of mRNA function and regulation.
• Current methods for analyzing poly(A) tails can be limited in scope or resolution.

Purpose of the Study:

• To present a detailed protocol for Nanopore 3' end-capture sequencing (Nano3P-seq).
• To introduce PolyTailor software for analyzing poly(A) tail length and composition.
• To enable single-molecule resolution analysis of RNA tail dynamics across various RNA types.

Main Methods:

• Development and validation of the Nano3P-seq protocol using Nanopore sequencing.
• Utilizing a template switching-based approach for cDNA sequencing from the 3' end.
• Employing the PolyTailor software for data analysis and prediction of tail characteristics.

Main Results:

• Nano3P-seq accurately estimates transcript abundances, poly(A) tail lengths, and composition.
• The method successfully analyzes coding and noncoding RNAs, including mRNAs, snoRNAs, and rRNAs.
• The protocol is compatible with R10.4 flow cells and can be completed within a day.

Conclusions:

• Nano3P-seq offers a comprehensive and reproducible method for studying RNA tail dynamics.
• The protocol is adaptable to various RNA sample preparation methods.
• This technique enhances the study of transcriptome-wide tail heterogeneity.