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RNA Biol. 2013 Apr;10(4):502-15. doi: 10.4161/rna.24284. Epub 2013 Apr 1.

Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material.

Author information

1
Biotechnology and Bioengineering, Sandia National Laboratories, Livermore, CA, USA.

Abstract

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

KEYWORDS:

Next generation sequencing (NGS); RNA-Seq; library preparation; quantitative PCR (qPCR); second generation sequencing (SGS)

PMID:
23558773
PMCID:
PMC3710357
DOI:
10.4161/rna.24284
[Indexed for MEDLINE]
Free PMC Article
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