Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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

Nature Protocols volume 7, pages 542–561 (2012)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Workflow of CAGE library preparation.
Figure 2: Strategies for eliminating noncapped biotinylated molecules.
Figure 3: Linker dimer elimination strategies.
Figure 4: Cap-trapped cDNA size distribution.
Figure 5: Measurement of PCR products.
Figure 6: Scatter plot of cluster expression between two biological replicates (K562 whole cell).

Similar content being viewed by others

References

  1. Schena, M., Shalon, D., Davis, R.W. & Brown, P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470 (1995).

    Article  CAS  Google Scholar 

  2. Cheng, J. et al. Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science 308, 1149–1154 (2005).

    Article  CAS  Google Scholar 

  3. Forrest, A.R. & Carninci, P. Whole genome transcriptome analysis. RNA Biol. 6, 107–112 (2009).

    Article  CAS  Google Scholar 

  4. Velculescu, V.E., Zhang, L., Vogelstein, B. & Kinzler, K.W. Serial analysis of gene expression. Science 270, 484–487 (1995).

    Article  CAS  Google Scholar 

  5. Kodzius, R. et al. CAGE: cap analysis of gene expression. Nat. Methods 3, 211–222 (2006).

    Article  CAS  Google Scholar 

  6. Kanamori-Katayama, M. et al. Unamplified cap analysis of gene expression on a single-molecule sequencer. Genome Res. 21, 1150–1159 (2011).

    Article  CAS  Google Scholar 

  7. Kawaji, H. et al. Dynamic usage of transcription start sites within core promoters. Genome Biol. 7, R118 (2006).

    Article  Google Scholar 

  8. Ponjavic, J. et al. Transcriptional and structural impact of TATA-initiation site spacing in mammalian core promoters. Genome Biol. 7, R78 (2006).

    Article  Google Scholar 

  9. Frith, M.C. et al. Evolutionary turnover of mammalian transcription start sites. Genome Res. 16, 713–2 (2006).

    Article  CAS  Google Scholar 

  10. Hoskins, R.A. et al. Genome-wide analysis of promoter architecture in Drosophila melanogaster. Genome Res. 21, 182–192 (2011).

    Article  CAS  Google Scholar 

  11. Carninci, P. et al. Genome-wide analysis of mammalian promoter architecture and evolution. Nat. Genet. 38, 626–635 (2006).

    Article  CAS  Google Scholar 

  12. Gustincich, S. et al. The complexity of the mammalian transcriptome. J. Physiol. 575, 321–332 (2006).

    Article  CAS  Google Scholar 

  13. Vitezic, M. et al. Building promoter aware transcriptional regulatory networks using siRNA perturbation and deepCAGE. Nucleic Acids Res. 38, 8141–8148 (2010).

    Article  CAS  Google Scholar 

  14. Frith, M.C. et al. A code for transcription initiation in mammalian genomes. Genome Res. 18, 1–12 (2008).

    Article  CAS  Google Scholar 

  15. Suzuki, H. et al. The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line. Nat. Genet. 41, 553–562 (2009).

    Article  CAS  Google Scholar 

  16. Faulkner, G.J. et al. The regulated retrotransposon transcriptome of mammalian cells. Nat. Genet. 41, 563–571 (2009).

    Article  CAS  Google Scholar 

  17. Hestand, M.S. et al. Tissue-specific transcript annotation and expression profiling with complementary next-generation sequencing technologies. Nucleic Acids Res. 38, e165 (2010).

    Article  Google Scholar 

  18. Wei, C.L. et al. 5′ Long serial analysis of gene expression (LongSAGE) and 3′ LongSAGE for transcriptome characterization and genome annotation. Proc. Natl. Acad. Sci. USA. 101, 11701–11706 (2004).

    Article  CAS  Google Scholar 

  19. Valen, E. et al. Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE. Genome Res. 19, 255–265 (2009).

    Article  CAS  Google Scholar 

  20. Shiraki, T. et al. Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proc. Natl. Acad. Sci. USA 100, 15776–15781 (2003).

    Article  CAS  Google Scholar 

  21. Myers, R.M. et al. A user's guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol. 9, e1001046 (2011).

    Article  CAS  Google Scholar 

  22. Takahashi, H., Kato, S., Murata, M. & Carninci, P. CAGE (Cap Analysis of Gene Expression): a protocol for the detection of promoter and transcriptional networks. Methods Mol. Biol. 786, 181–200 (2012).

    Article  CAS  Google Scholar 

  23. Carninci, P. et al. The transcriptional landscape of the mammalian genome. Science 309, 1559–1563 (2005).

    Article  CAS  Google Scholar 

  24. Carninci, P. et al. Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. Proc. Natl. Acad. Sci. USA 95, 520–524 (1998).

    Article  CAS  Google Scholar 

  25. Carninci, P., Shiraki, T., Mizuno, Y., Muramatsu, M. & Hayashizaki, Y. Extra-long first-strand cDNA synthesis. Biotechniques 32, 984–985 (2002).

    Article  CAS  Google Scholar 

  26. Carninci, P. et al. High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics 37, 327–336 (1996).

    Article  CAS  Google Scholar 

  27. Shibata, K. et al. RIKEN integrated sequence analysis (RISA) system—384-format sequencing pipeline with 384 multicapillary sequencer. Genome Res. 10, 1757–1771 (2000).

    Article  CAS  Google Scholar 

  28. Plessy, C. et al. Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan. Nat. Methods 7, 528–534 (2010).

    Article  CAS  Google Scholar 

  29. Maeda, N. et al. Development of a DNA barcode tagging method for monitoring dynamic changes in gene expression by using an ultra high-throughput sequencer. Biotechniques 45, 95–97 (2008).

    Article  CAS  Google Scholar 

  30. Janscak, P., Sandmeier, U., Szczelkun, M.D. & Bickle, T.A. Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I. J. Mol. Biol. 306, 417–431 (2001).

    Article  CAS  Google Scholar 

  31. Raghavendra, N.K. & Rao, D.N. Exogenous AdoMet and its analogue sinefungin differentially influence DNA cleavage by R.EcoP15I–usefulness in SAGE. Biochem. Biophys. Res. Commun. 334, 803–811 (2005).

    Article  CAS  Google Scholar 

  32. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).

    Article  CAS  Google Scholar 

  33. Lassmann, T., Hayashizaki, Y. & Daub, C.O. TagDust—a program to eliminate artifacts from next generation sequencing data. Bioinformatics 25, 2839–2840 (2009).

    Article  CAS  Google Scholar 

  34. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  Google Scholar 

  35. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  36. Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  Google Scholar 

  37. Fujita, P.A. et al. The UCSC Genome Browser database: update 2011. Nucleic Acids Res. 39, D876–D882 (2011).

    Article  CAS  Google Scholar 

  38. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    Article  CAS  Google Scholar 

  39. Carninci, P. Cap-analysis Gene Expression (CAGE): The Science of Decoding Gene Transcription (Pan Stanford, 2010).

  40. Li, Q., Brown, J.B., Huang, H. & Bickel, P.J. Measuring reproducibility of high-throughput experiments. Ann. Appl. Stat. 5, 1752–1779 (2011).

    Article  Google Scholar 

Download references

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

Authors and Affiliations

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

    Hazuki Takahashi, Timo Lassmann, Mitsuyoshi Murata & Piero Carninci

Authors
  1. Hazuki Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Timo Lassmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mitsuyoshi Murata
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Piero Carninci
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Piero Carninci.

Ethics declarations

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.

Supplementary information

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. (PNG 652 kb)

Supplementary Data 1

The make_ctss script, which is used to cluster the CTSS (Step 65). (TXT 1 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Takahashi, H., Lassmann, T., Murata, M. et al. 5′ end–centered expression profiling using cap-analysis gene expression and next-generation sequencing. Nat Protoc 7, 542–561 (2012). https://doi.org/10.1038/nprot.2012.005

Download citation

  • Published:

  • Issue Date:

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

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing