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5′ end–centered expression profiling using cap-analysis gene expression and next-generation sequencing

Nature Protocols volume 7, pages 542561 (2012) | Download Citation

Abstract

Cap-analysis gene expression (CAGE) provides accurate high-throughput measurement of RNA expression. CAGE allows mapping of all the initiation sites of both capped coding and noncoding RNAs. In addition, transcriptional start sites within promoters are characterized at single-nucleotide resolution. The latter allows the regulatory inputs driving gene expression to be studied, which in turn enables the construction of transcriptional networks. Here we provide an optimized protocol for the construction of CAGE libraries on the basis of the preparation of 27-nt-long tags corresponding to initial bases at the 5′ ends of capped RNAs. We have optimized the methods using simple steps based on filtration, which altogether takes 4 d to complete. The CAGE tags can be readily sequenced with Illumina sequencers, and upon modification they are also amenable to sequencing using other platforms.

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Acknowledgements

This work was founded by a Research Grant for the RIKEN Omics Science Center from the Japanese Ministry of Education, Culture, Sports, Science and Technology (to Y.H.) This project was also supported by the US National Human Genome Research Institute grant no. U54 HG004557. We thank S. Kato for experimental support, J. Severin for the genome browser, the RIKEN Genome Network Analysis Service for sequencing and basic bioinformatics analysis, and all our colleagues at the Omics Science Center for valuable feedback during the development of the methodology.

Author information

Affiliations

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

    • Hazuki Takahashi
    • , Timo Lassmann
    • , Mitsuyoshi Murata
    •  & Piero Carninci

Authors

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Contributions

H.T. performed most experiments. M.M. performed the background reduction experiment. T.L. performed computations analysis. H.T. and P.C. wrote the manuscript. P.C. designed the project.

Competing interests

P.C. is an inventor on various patents owned by RIKEN and Dnaform on the Cap-trapper technology, full-length cDNA cloning technologies and the CAGE technology.

Corresponding author

Correspondence to Piero Carninci.

Supplementary information

Image files

  1. 1.

    Supplementary Fig. 1

    Oligo-dT priming enhances the capture of CAGE tags on exons and 3′ UTRs.  CAGE libraries made from THP-1 cells. Data was displayed with the ZENBU genome browser (J. Severin, unpublished data). (a) The Actin beta gene is transcribed from right to left (violet arrow) on chromosome 7. (b) GAPDH gene is transcribed from left to right (green arrow) on chromosome 12. CAGE libraries were primed RT reaction with (1) random and oligodT (ratio 4:1) primers. (2) oligodT primers only and (3) random primers only. Both panels indicate that oligodT primers could enhance the capture of transcripts on 3′ exons and on internal exons, compared to random primer alone.

Text files

  1. 1.

    Supplementary Data 1

    The make_ctss script, which is used to cluster the CTSS (Step 65).

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DOI

https://doi.org/10.1038/nprot.2012.005

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