A Rapid End-to-End Workflow for Preparing rRNA-depleted and Directional Plant RNA-Seq Libraries

Massively parallel sequencing of cDNA libraries (RNA-Seq) is now routinely used for transcript profiling and analysis of novel transcripts, novel isoforms, alternative splice sites, rare transcripts and cSNPs, compared to microarrays. However, some methods for preparing rRNA-depleted RNA-Seq libraries are generally time-consuming with multiple cleanup steps, requiring about 2 days with significant hands-on time, which can be costly.

Here, we present an efficient “single-pass” plant rRNA removal method (Ribo-Zero™ technology) combined with the rapid and user-friendly ScriptSeq™ RNA-Seq library preparation method in an end-to-end workflow (ScriptSeq^TM Complete) for preparing directional plant cDNA libraries from nanogram amounts of total RNA with ~98% strandedness in <1 day (5-6 hours). NGS libraries contain <2 % of reads that map to rRNA sequences (cytoplasmic, chloroplast and mitochondrial) compared to ~74% (Arabidopsis thaliana) that map to mRNA sequences, while maintaining representation of coding and noncoding transcripts. This reduction in rRNA sequence reads improves sequence depth and coverage of mRNA, and increases the percentage of uniquely mapped reads.