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Digital transcriptome profiling using selective hexamer priming for cDNA synthesis

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Abstract

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.

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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.

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Acknowledgements

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.

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Authors and Affiliations

  1. Departments of Molecular Informatics, a wholly owned subsidiary of Merck and Co., Inc., Seattle, Washington, USA

    Christopher D Armour, John C Castle, Ronghua Chen, Tomas Babak, Patrick Loerch, Jyoti K Shah, Carol A Rohl, Jason M Johnson & Christopher K Raymond

  2. Scientific Computing, Rosetta Inpharmatics LLC, a wholly owned subsidiary of Merck and Co., Inc., Seattle, Washington, USA

    Stuart Jackson & John Dey

Authors
  1. Christopher D Armour
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  2. John C Castle
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  3. Ronghua Chen
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  4. Tomas Babak
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  5. Patrick Loerch
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  6. Stuart Jackson
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  7. Jyoti K Shah
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  8. John Dey
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  9. Carol A Rohl
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  10. Jason M Johnson
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  11. Christopher K Raymond
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Contributions

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.

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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)

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Armour, C., Castle, J., Chen, R. et al. Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nat Methods 6, 647–649 (2009). https://doi.org/10.1038/nmeth.1360

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  • DOI: https://doi.org/10.1038/nmeth.1360

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