The Use of Nanopore Sequencing to Analyze the Chloroplast Transcriptome Part I: Library Preparation.

Global understanding of plastid gene expression has always been impaired by its complexity. RNA splicing, editing, and intercistronic processing create multiple transcripts isoforms that can hardly be resolved using traditional molecular biology techniques. During the last decade, the wide adoption of RNA-seq-based techniques has, however, allowed an unprecedented understanding of all the different steps of chloroplast gene expression, from transcription to translation. Current strategies are nonetheless unable to identify and quantify full length transcripts isoforms, a limitation that can now be overcome using Nanopore Sequencing. We here provide a complete protocol to produce, from total leaf RNA, cDNA libraries ready for Nanopore sequencing. While most Nanopore protocols take advantage of the mRNA polyA tail we here first ligate an RNA adapter to the 3' ends of the RNAs and use it to initiate the template switching reverse transcription. The cDNA is then prepared and indexed for use with the regular Oxford Nanopore v14 chemistry. This protocol is of particular interest to researchers willing to simultaneously study the multiple post-transcriptional processes prevalent in the chloroplast.