Abstract

INTRODUCTION Conventional Illumina RNA-Seq does not have the resolution to decode the complex eukaryote transcriptome due to the lack of RNA polarity information. Strand-specific RNA sequencing (ssRNA-Seq) can overcome these limitations and as such is better suited for genome annotation, de novo transcriptome assembly, and accurate digital gene expression analysis. This protocol describes a simple and robust method to generate ssRNA-Seq libraries for the Illumina sequencing platform. It has significantly increased the throughput to 96 libraries in a two-day preparation while simultaneously lowering the reagent costs to below ten dollars per library. It is compatible with both single-read and paired-end multiplex sequencing and, most importantly, its data can also be used with existing conventional RNA-Seq data. This is a significant advantage, because it enables researchers to switch to ssRNA-Seq even if a large amount of data has already been generated by the nonstrand specific methods.