UNIT 4.21 Next-Generation Sequencing RNA-Seq Library Construction

  1. Jessica Podnar,
  2. Heather Deiderick,
  3. Gabriella Huerta,
  4. Scott Hunicke-Smith

Published Online: 14 APR 2014

DOI: 10.1002/0471142727.mb0421s106

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Podnar, J., Deiderick, H., Huerta, G. and Hunicke-Smith, S. 2014. Next-Generation Sequencing RNA-Seq Library Construction. Current Protocols in Molecular Biology. 106:II:4.21:4.21.1–4.21.19.

Author Information

  1. Genomic Sequencing and Analysis Facility, University of Texas at Austin, Austin, Texas

Publication History

  1. Published Online: 14 APR 2014


This unit presents protocols for construction of next-generation sequencing (NGS) directional RNA sequencing libraries for the Illumina HiSeq and MiSeq from a wide variety of input RNA sources. The protocols are based on the New England Biolabs (NEB) small RNA library preparation set for Illumina, although similar kits exist from different vendors. The protocol preserves the orientation of the original RNA in the final sequencing library, enabling strand-specific analysis of the resulting data. These libraries have been used for differential gene expression analysis and small RNA discovery and are currently being tested for de novo transcriptome assembly. The protocol is robust and applicable to a broad range of RNA input types and RNA quality, making it ideal for high-throughput laboratories. Curr. Protoc. Mol. Biol. 106:4.21.1-4.21.19. © 2014 by John Wiley & Sons, Inc.


  • RNA-Seq;
  • NGS;
  • library construction;
  • transcriptome;
  • strand-specific;
  • gene expression