A new method for stranded whole transcriptome RNA-seq

David F.B. Miller, Pearlly S. Yan, Aaron Buechlein, Benjamin A. Rodriguez, Ayse S. Yilmaz, Shokhi Goel, Hai Lin, Bridgette Collins-Burow, Lyndsay V. Rhodes, Chris Braun, Sunila Pradeep, Rajesha Rupaimoole, Mehmet Dalkilic, Anil K. Sood, Matthew E. Burow, Haixu Tang, Tim H. Huang, Yunlong Liu, Douglas B. Rusch, Kenneth P. Nephew

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).

Original languageEnglish (US)
Pages (from-to)126-134
Number of pages9
JournalMethods
Volume63
Issue number2
DOIs
StatePublished - Sep 15 2013

Keywords

  • Duplex-specific nuclease
  • Gene expression
  • RNA-seq
  • Transcriptome

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Miller, D. F. B., Yan, P. S., Buechlein, A., Rodriguez, B. A., Yilmaz, A. S., Goel, S., Lin, H., Collins-Burow, B., Rhodes, L. V., Braun, C., Pradeep, S., Rupaimoole, R., Dalkilic, M., Sood, A. K., Burow, M. E., Tang, H., Huang, T. H., Liu, Y., Rusch, D. B., & Nephew, K. P. (2013). A new method for stranded whole transcriptome RNA-seq. Methods, 63(2), 126-134. https://doi.org/10.1016/j.ymeth.2013.03.023