Unit

UNIT 4.12 Construction of Normalized RNA-seq Libraries for Next-Generation Sequencing Using the Crab Duplex-Specific Nuclease

  1. Danos C. Christodoulou,
  2. Joshua M. Gorham,
  3. Daniel S. Herman,
  4. J.G. Seidman

Published Online: 1 APR 2011

DOI: 10.1002/0471142727.mb0412s94

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Christodoulou, D. C., Gorham, J. M., Herman, D. S. and Seidman, J. 2011. Construction of Normalized RNA-seq Libraries for Next-Generation Sequencing Using the Crab Duplex-Specific Nuclease. Current Protocols in Molecular Biology. 94:II:4.12:4.12.1–4.12.11.

Author Information

  1. Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 APR 2011
  2. Published Print: APR 2011

Abstract

RNA-seq is a method for studying the transcriptome of cells or tissues by massively parallel sequencing of tens of millions of short DNA fragments. However, the broad dynamic range of gene expression levels, which span more than five orders of magnitude, necessitates considerable over-sequencing to characterize low-abundance RNAs at sufficient depth. Here, we describe a method that enables efficient sequencing of low-abundance RNAs by normalizing or reducing the range spanned by the most abundant RNA species to the least abundant RNA species. This normalization is achieved using an approach that was developed for generating expressed sequence tag (EST) libraries that uses the crab duplex-specific nuclease and exploits the kinetics of DNA annealing. That is, double-stranded cDNA is denatured, then allowed to partially re-anneal, and the most abundant species, which re-anneal most rapidly, are digested with crab duplex-specific nuclease. This procedure substantially decreases the proportion of sequence reads from highly expressed RNAs, facilitating assessment of the full spectrum of the sequence and structure of transcriptomes. Curr. Protoc. Mol. Biol. 94:4.12.1-4.12.11. © 2011 by John Wiley & Sons, Inc.

Keywords:

  • RNA-seq;
  • library normalization;
  • crab duplex nuclease;
  • high-throughput DNA sequencing