High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation
- Silin Zhong1,2,5,
- Je-Gun Joung1,
- Yi Zheng1,
- Yun-ru Chen1,
- Bao Liu2,
- Ying Shao3,
- Jenny Z. Xiang3,
- Zhangjun Fei1,4,5 and
- James J. Giovannoni1,4,5
- 1Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA
- 2Institute of Genetics and Cytology, Northeast Normal University, Changchun, 130024, China
- 3Weill Medical College, Cornell University, New York, NY 10021, USA
- 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
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↵5 Corresponding authors (jjg33{at}cornell.edu, zf25{at}cornell.edu, and sz284{at}cornell.edu)
- Published by Cold Spring Harbor Laboratory Press