Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Digital transcriptome profiling using selective hexamer priming for cDNA synthesis


We developed a procedure for the preparation of whole transcriptome cDNA libraries depleted of ribosomal RNA from only 1 μg of total RNA. The method relies on a collection of short, computationally selected oligonucleotides, called 'not-so-random' (NSR) primers, to obtain full-length, strand-specific representation of nonribosomal RNA transcripts. In this study we validated the technique by profiling human whole brain and universal human reference RNA using ultra-high-throughput sequencing.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Construction of NSR-primed whole transcriptome cDNA libraries.
Figure 2: Detection of poly(A) noncoding RNAs in NSR-primed cDNA libraries.
Figure 3: Classification of global transcriptional activity.

Accession codes




  1. Cloonan, N. et al. Nat. Methods 5, 613–619 (2008).

    Article  CAS  Google Scholar 

  2. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Nat. Methods 5, 621–628 (2008).

    Article  CAS  Google Scholar 

  3. Morin, R. et al. Biotechniques 45, 81–94 (2008).

    Article  CAS  Google Scholar 

  4. Yoder-Himes, D.R. et al. Proc. Natl. Acad. Sci. USA 106, 3976–3981 (2009).

    Article  CAS  Google Scholar 

  5. Raymond, C.K., Roberts, B.S., Garrett-Engele, P., Lim, L.P. & Johnson, J.M. RNA 11, 1737–1744 (2005).

    Article  CAS  Google Scholar 

  6. Pruitt, K.D., Tatusova, T. & Maglott, D.R. Nucleic Acids Res. 33, D501–D504 (2005).

    Article  CAS  Google Scholar 

  7. Shi, L. et al. Nat. Biotechnol. 24, 1151–1161 (2006).

    Article  CAS  Google Scholar 

  8. Wang, E.T. et al. Nature 456, 470–476 (2008).

    Article  CAS  Google Scholar 

  9. Wu, J.Q. et al. Genome Biol. 9, R3 (2008).

    Article  Google Scholar 

  10. Cavaille, J. et al. Proc. Natl. Acad. Sci. USA 97, 14311–14316 (2000).

    Article  CAS  Google Scholar 

  11. Katayama, S. et al. Science 309, 1564–1566 (2005).

    Article  Google Scholar 

  12. Gonzalez, J.M. & Robb, F.T. J. Microbiol. Methods 71, 288–291 (2007).

    Article  CAS  Google Scholar 

  13. Guttman, M. et al. Nature 458, 223–227 (2009).

    Article  CAS  Google Scholar 

  14. Amaral, P.P., Dinger, M.E., Mercer, T.R. & Mattick, J.S. Science 319, 1787–1789 (2008).

    Article  CAS  Google Scholar 

  15. Babak, T. et al. Curr. Biol. 18, 1735–1741 (2008).

    Article  CAS  Google Scholar 

Download references


We thank T. Fare, L. Lim, D. Haynor, P. Lum and E. Schadt for valuable input, M. Biery and H. Bouzek for technical assistance, and G. Schroth and M. Schlador for advice.

Author information

Authors and Affiliations



C.D.A., J.C.C. and C.K.R. contributed to the conceptual development and experimental design. C.D.A. and C.K.R. constructed libraries and generated sequencing data. S.J. and J.D. analyzed images and managed the base calling pipeline. J.C.C., R.C., T.B., P.L. and J.K.S. performed sequence alignments and genome analysis. C.A.R. and J.M.J. provided analysis support and project management. C.D.A. and C.K.R. prepared the manuscript.

Corresponding author

Correspondence to Christopher D Armour.

Ethics declarations

Competing interests

C.D.A., J.C.C., R.C., T.B., P.L., S.J., J.K.S., J.D., C.A.R., J.M.J. and C.K.R. are paid employees of Merck and Company, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 4 (PDF 249 kb)

Supplementary Table 1

First strand NSR primer sequences. (XLS 53 kb)

Supplementary Table 2

Second strand NSR primer sequences. (XLS 68 kb)

Supplementary Table 3

Non-coding RNA expression levels in NSR libraries. (XLS 64 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Armour, C., Castle, J., Chen, R. et al. Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nat Methods 6, 647–649 (2009).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing