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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Change history
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.
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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.
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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.
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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.
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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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DOI: https://doi.org/10.1038/nmeth.1470
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