Methods and Protocols (Mar 2023)

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

  • Daniela Cordeiro,
  • Alexandra Camelo,
  • Ana Carolina Pedrosa,
  • Inês Brandão,
  • Jorge Canhoto,
  • Christophe Espírito Santo,
  • Sandra Correia

DOI
https://doi.org/10.3390/mps6020031
Journal volume & issue
Vol. 6, no. 2
p. 31

Abstract

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Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.

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