Digital transcriptome profiling using selective hexamer priming for cDNA synthesis (original) (raw)

• Brief Communication
• Published: 09 August 2009
• John C Castle1,
• Ronghua Chen1,
• Tomas Babak1,
• Patrick Loerch1,
• Stuart Jackson2,
• Jyoti K Shah1,
• John Dey2,
• Carol A Rohl1,
• Jason M Johnson1 &
• …
• Christopher K Raymond1

Nature Methods volume 6, pages 647–649 (2009)Cite this article

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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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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 toChristopher 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.

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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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• Received: 23 March 2009
• Accepted: 09 July 2009
• Published: 09 August 2009
• Issue Date: September 2009
• DOI: https://doi.org/10.1038/nmeth.1360

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