Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue

Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from polyadenylated mRNA for transcriptome analysis. Both high-quality and high-throughput protocol options include steps of mRNA enrichment through magnetic bead-enabled precipitation of the poly-A tail, cDNA synthesis, and then fragmentation and adapter addition simultaneously through Tn5-mediated 'tagmentation'. All steps of the protocols have been validated with Arabidopsis thaliana leaf and seedling tissues and streamlined to work together, with minimal cost in money and time, thus intended to provide a beginner-friendly start-to-finish RNA-seq library preparation for transcriptome analysis.