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Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan

Nature Methods volume 7, pages 528–534 (2010)Cite this article

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Abstract

Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5′ ends that vary in size. Methods to identify the 5′ ends of transcripts will facilitate the discovery of new promoters and 5′ ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano–cap analysis of gene expression (nanoCAGE), a method that captures the 5′ ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5′ ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome.

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Figure 1: Experimental outline of the nanoCAGE and CAGEscan protocols.
Figure 2: nanoCAGE specifically captures capped 5′ ends.
Figure 3: Promotome-transcriptome analysis with CAGEscan.
Figure 4: CAGEscan connects promoters and downstream sequences.
Figure 5: Expressed repeat elements surveyed by CAGEscan.

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Change history

  • (2010年10月06日 追記)

    06 October 2010

    In the version of this article initially published, the concentration of the PCR primers for CAGEscan cDNA amplification and CAGEscan cDNA sequencing were erroneously given as millimolar instead of nanomolar. The error has been corrected in the HTML and PDF versions of the article.

    (追記ここまで)

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Acknowledgements

This work was funded by a grant of the 6th Framework of the European Union commission to the Neuro Functional Genomics consortium, by a grant of the 7th Framework to P.C. and S.G. (Dopaminet), a Grant-in-Aids for Scientific Research (A) 20241047 for P.C. and a Research Grant for RIKEN Omics Science Center from the Japanese Ministry of Education, Culture, Sports, Science and Technology to Y.H. This project was also partially supported by the U.S. National Human Genome Research Institute grant U54 HG004557. C.P. was supported by the Japanese Society for the Promotion of Science long-term fellowship P05880. S.G. was funded by a career developmental award from The Giovanni Armenise-Harvard Foundation. We thank A. Forrest for critical discussions, M. Josserand for experimental assistance and RIKEN Genome Network Analysis Service for data production.

Author information

Author notes

  1. Signe Olivarius & Philipp Kapranov

    Present address: Present addresses: Molecular Evolution Group, Department of Biology, University of Copenhagen, Copenhagen, Denmark (S.O.) and Helicos BioSciences Corporation, Cambridge, Massachusetts, USA (P.K.).,

  2. Charles Plessy, Nicolas Bertin, Hazuki Takahashi and Roberto Simone: These authors contributed equally to this work.

Authors and Affiliations

  1. RIKEN Yokohama Institute, Omics Science Center, Yokohama, Japan

    Charles Plessy, Nicolas Bertin, Hazuki Takahashi, Md Salimullah, Timo Lassmann, Morana Vitezic, Jessica Severin, Signe Olivarius, Jun Kawai, Carsten O Daub, Yoshihide Hayashizaki & Piero Carninci

  2. Sector of Neurobiology, International School for Advanced Studies, Trieste, Italy

    Roberto Simone, Dejan Lazarevic & Stefano Gustincich

  3. Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden

    Morana Vitezic

  4. Dulbecco Telethon Institute, Epigenetics and Genome Reprogramming Laboratory, Santa Lucia Foundation at European Brain Research Institute, Rome, Italy

    Nadine Hornig & Valerio Orlando

  5. Affymetrix Inc., Santa Clara, California, USA

    Ian Bell, Hui Gao, Jacqueline Dumais & Philipp Kapranov

  6. Cold Spring Harbor Laboratory, Genome Center, Woodbury, New York, USA

    Huaien Wang & Carrie A Davis

  7. Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Thomas R Gingeras

Authors
  1. Charles Plessy
  2. Nicolas Bertin
  3. Hazuki Takahashi
  4. Roberto Simone
  5. Md Salimullah
  6. Timo Lassmann
  7. Morana Vitezic
  8. Jessica Severin
  9. Signe Olivarius
  10. Dejan Lazarevic
  11. Nadine Hornig
  12. Valerio Orlando
  13. Ian Bell
  14. Hui Gao
  15. Jacqueline Dumais
  16. Philipp Kapranov
  17. Huaien Wang
  18. Carrie A Davis
  19. Thomas R Gingeras
  20. Jun Kawai
  21. Carsten O Daub
  22. Yoshihide Hayashizaki
  23. Stefano Gustincich
  24. Piero Carninci

Contributions

C.P., R.S. and P.C. conceived the nanoCAGE technology. C.P. and P.C. conceived the CAGEscan technology. C.P., H.T., R.S., M.S. and S.O. designed and performed the experiments. C.P., N.B., H.T., T.L. and M.V. analyzed data and interpreted results. C.P., N.B., S.G. and P.C. supervised the study. D.L., N.H., V.O., I.B., H.G., J.D., P.K., H.W., C.A.D. and T.R.G. provided material. J.S. provided software. J.K., Y.H., S.G. and P.C. provided salary support. P.C., N.B., H.T. and M.V. drafted the text and figures. C.P., N.B., T.L., C.O.D. and P.C. edited the text and figures. General correspondence should be addressed to S.G. or P.C.; correspondence about experiments, to C.P.; and correspondence about bioinformatics, to N.B.

Corresponding authors

Correspondence to Charles Plessy, Nicolas Bertin, Stefano Gustincich or Piero Carninci.

Ethics declarations

Competing interests

C.P., P.C. and R.S. are inventors of the Japanese patent application held by RIKEN on the moderately suppressive PCR step of the nanoCAGE protocol. P.K. is currently an employee of Helicos BioSciences.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–8 (PDF 2743 kb)

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Plessy, C., Bertin, N., Takahashi, H. et al. Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan. Nat Methods 7, 528–534 (2010). https://doi.org/10.1038/nmeth.1470

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