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NAP-seq for full-length noncapped RNA sequencing.

Shurong Liu1, Junhong Huang1, Lianghu Qu2

  • 1MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Pharmaceutical Functional Genes, Innovation Center for Evolutionary Synthetic Biology, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.

Nature Protocols
|September 17, 2025
PubMed
Summary
This summary is machine-generated.

Noncapped RNAs (napRNAs) are abundant but hard to study. NAP-seq is a new method to sequence full-length napRNAs, aiding the discovery of novel regulatory RNAs and RNA biogenesis pathways.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biochemistry

Background:

  • The mammalian genome extensively transcribes noncapped RNAs (napRNAs).
  • napRNAs regulate biological processes and RNA biogenesis but are challenging to identify due to their heterogeneity.
  • Existing methods struggle with napRNA's diverse lengths, modifications, and structures.

Purpose of the Study:

  • To present the principles and detailed procedures of the napRNA sequencing (NAP-seq) technique.
  • To enable comprehensive identification of full-length napRNAs across various cell types.
  • To overcome challenges in napRNA discovery and characterization.

Main Methods:

  • NAP-seq utilizes T4 polynucleotide kinase pretreatment for standardized RNA termini.
  • Size-selection and RNase H depletion enrich for long, low-abundance RNAs.
  • Custom adapters with random barcodes, dual-platform sequencing (Oxford Nanopore and Illumina), and optimized cDNA synthesis ensure single-nucleotide resolution and minimize biases.

Main Results:

  • NAP-seq allows for the identification of full-length napRNAs with various terminal modifications at single-nucleotide resolution.
  • The method effectively enriches for low-abundance, long RNAs.
  • The entire workflow, from library prep to analysis, can be completed within 8 days.

Conclusions:

  • NAP-seq provides a robust approach for discovering novel noncoding RNAs with regulatory roles.
  • This technique facilitates the investigation of RNA biogenesis in diverse biological contexts.
  • NAP-seq overcomes previous limitations in napRNA identification and characterization.