Protocol

High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation

  1. James J. Giovannoni1,4,5
  1. 1Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA
  2. 2Institute of Genetics and Cytology, Northeast Normal University, Changchun, 130024, China
  3. 3Weill Medical College, Cornell University, New York, NY 10021, USA
  4. 4U.S. Department of Agriculture/Agriculture Research Service, Plant, Soil, and Nutrition Laboratory, Ithaca, NY 14853, USA

    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.

    Footnotes

    • 5 Corresponding authors (jjg33{at}cornell.edu, zf25{at}cornell.edu, and sz284{at}cornell.edu)

    Responses to this article

    | Table of Contents