Identification of regulatory links between transcription and RNA processing with long-read sequencing
Carlos Alfonso-Gonzalez1, Laura Arrigoni2, Hasan Can Ozbulut3
1Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany; Faculty of Biology, Albert Ludwig University, 79104 Freiburg, Germany; International Max Planck Research School for Molecular and Cellular Biology (IMPRS- MCB), 79108 Freiburg, Germany.
STAR Protocols
|September 21, 2023
Summary
This study details a protocol for sequencing full-length messenger RNA (mRNA) isoforms using Oxford Nanopore long-read technology. The method links transcription initiation to RNA processing events for comprehensive transcript analysis.
Area of Science:
- Molecular Biology
- Genomics
- Bioinformatics
Background:
- Accurate sequencing of full-length mRNA isoforms is crucial for understanding gene expression and regulation.
- Existing methods may have limitations in capturing complete transcript structures and linking transcription to processing.
Purpose of the Study:
- To present a detailed protocol for sequencing full-length mRNA isoforms using Oxford Nanopore long-read sequencing.
- To enable the identification of connections between transcription initiation and co-transcriptional RNA processing.
Main Methods:
- Poly(A) RNA isolation and library preparation.
- Complementary DNA (cDNA) size selection.
- Oxford Nanopore sequencing, data processing, and computational analysis for exon coupling and end assignment.
Main Results:
- A comprehensive protocol for full-length mRNA isoform sequencing.
- Identification of exon couplings and assignment of mRNA 5' and 3' ends.
- Framework to link transcription initiation with RNA processing events.
Conclusions:
- The developed protocol facilitates detailed analysis of mRNA isoforms.
- This approach enhances the understanding of the interplay between transcription and RNA processing.
- Enables comprehensive transcriptomic studies using long-read sequencing technology.
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