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An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing.

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Methods and Protocols
|March 24, 2023
PubMed
Summary

This study presents a new protocol for preparing cDNA libraries from woody plant tissues for long-read sequencing. This method overcomes challenges, enabling comprehensive plant transcriptome analysis.

Keywords:
MinIONOxford Nanopore Technologies®SolanaceaeSolanum betaceumamplification-free protocoldirect cDNA sequencinghigh-throughput sequencingpoly(A) RNAtranscriptomewoody plant

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Area of Science:

  • Plant biology
  • Genomics
  • Molecular biology

Background:

  • Long-read sequencing offers comprehensive transcriptome analysis, identifying full-length transcripts.
  • This method enhances gene annotation and expression studies, especially for non-model species.
  • Woody plant tissues present challenges for cDNA library preparation, hindering advanced sequencing.

Purpose of the Study:

  • To describe a detailed protocol for preparing cDNA libraries for high-throughput RNA sequencing using Oxford Nanopore Technologies.
  • To enable comprehensive transcriptome analysis in woody plants.

Main Methods:

  • Developed a protocol for cDNA library preparation from woody plant tissues.
  • Applied the protocol to two *Solanum betaceum* cell lines: embryogenic competent (EC) and non-embryogenic (NEC).
  • Prepared eight barcoded cDNA libraries for sequencing.

Main Results:

  • The protocol successfully yielded high-quality cDNA libraries from *Solanum betaceum*.
  • Sequencing data exhibited high mean quality scores.
  • Demonstrated the feasibility of long-read sequencing for plant transcriptome analysis.

Conclusions:

  • The described protocol facilitates cDNA library preparation from challenging woody plant tissues.
  • This method supports comprehensive plant transcriptome analysis using long-read sequencing.
  • Enables advanced gene annotation and expression studies in non-model plants.