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Nature Methods
  • Protocol
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CAGE: cap analysis of gene expression

Nature Methodsvolume 3, pages211–222 (2006)Cite this article

  • (+)-biotin hydrazide long-arm variant (Vector Laboratories)

  • Biotin solution D: 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sodiumN-lauroyl sarcosinate (Sarkosyl), 1.5% (wt/vol) freshly dissolved biotin

  • 1×BW buffer (Dynabeads M-280 streptavidin bind and wash buffer): 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA (pH 8.0), 1 M NaCl, 0.1 mg/ml bovine serum albumin (BSA)

  • Buffer PE (pH 7.7), ethanol-based wash buffer (Qiagen)

  • CL4B buffer: 1 mM EDTA, 10 mM Tris-HCl (pH 8.0), 100 μg/ml BSA, 0.1% sodium azide

  • CTAB-urea solution: 1% cetyltrimethylammonium bromide (CTAB; cationic detergent)8, 4 M urea, 50 mM Tris-HCl (pH 7.0), 1 mM EDTA

  • dNTP–5m-dCTP mix: solution containing dATP, dGTP, dTTP and 5-methyl-dCTP, each at a concentration of 10 mM

  • Dynabeads M-280 streptavidin (10 mg/ml, 6.7 × 108 beads/ml; Dynal)

  • 2× GC buffer I from TaKaRaLA Taq thermostable DNA polymerase kit (Takara)

  • LoTE (low-salt Tris-EDTA) buffer: 3 mM Tris-HCl (pH 7.5), 0.2 mM EDTA (pH 7.5)

  • Oligonucleotides (seeSupplementary Table 1 online)

  • Polyacrylamide gel elution buffer: 0.5 M NH4OAc, 10 mM Mg(OAc)2, 1 mM EDTA (pH 8.0), 0.1% SDS

  • Release buffer (prepare fresh): 50 mM NaOH, 5 mM EDTA

  • Sephacryl S-400 HR resin (Amersham Biosciences)

  • Sodium periodate (NaIO4), reagent grade (ICN)

  • Sorbitol-trehalose mix: 2:1 (vol/vol) of 4.9 M sorbitol and saturated solution (80%) ofD-(+)-trehalose (>99.5% by high performance liquid chromatography (HPLC); Fluka)

  • Streptavidin-agarose (Upstate)

  • 0.1× TE buffer: 1 mM Tris-HCl (pH 7.5), 0.1 mM EDTA

  • Wash buffer 1: 4.5 M NaCl, 50 mM EDTA (pH 8.0); wash buffer 2: 0.3 M NaCl, 1 mM EDTA; wash buffer 3: 0.4% SDS, 0.5 M NaOAc, 20 mM Tris-HCl (pH 8.5), 1 mM EDTA; wash buffer 4: 0.5 M NaOAc, 10 mM Tris-HCl (pH 8.5), 1 mM EDTA

(+)-biotin hydrazide long-arm variant (Vector Laboratories)

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Figure 1: Preparation of CAGE libraries.

References

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Acknowledgements

We are grateful to S. Kondo and A. Hasegawa for help with bioinformatics, and H. Sato, C. Kawazu, S. Kanagawa, M. Ohno, M. Murata, K. Nomura, Y. Tagami-Takeda and K. Hayashida for support in developing, producing and sequencing CAGE libraries. This protocol was developed with the support of the Genome Network Project, the Advanced and Innovational Research Program in Life Science and the Research Grant for RIKEN Genome Exploration Research Project, all from the Ministry of Education, Culture, Sports, Science and Technology, as well as the Strategic Programs for Research and Development of RIKEN. R.K. was supported by a fellowship from the European Union (FP5 INCO2 to Japan).

Author information

Author notes

  1. Rimantas Kodzius

    Present address: Vaxine Pty Ltd., Department of Diabetes and Endocrinology, Flinders Medical Centre, Bedford Park, Southern Australia, 5042, Australia

Authors and Affiliations

  1. Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), Yokohama Institute 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Kanagawa, Japan

    Rimantas Kodzius, Miki Kojima, Hiromi Nishiyori, Mari Nakamura, Shiro Fukuda, Michihira Tagami, Daisuke Sasaki, Kengo Imamura, Chikatoshi Kai, Yoshihide Hayashizaki & Piero Carninci

  2. Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa Wako, Saitama, 351-0198, Japan

    Rimantas Kodzius, Yoshihide Hayashizaki & Piero Carninci

  3. K.K. Dnaform, Tsukuba Branch, 3-1 Chuo 8-chome, Ami Machi, Inashiki Gun, 300-0332, Ibaraki, Japan

    Matthias Harbers

Authors
  1. Rimantas Kodzius

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  2. Miki Kojima

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  3. Hiromi Nishiyori

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  4. Mari Nakamura

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  5. Shiro Fukuda

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  6. Michihira Tagami

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  7. Daisuke Sasaki

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  8. Kengo Imamura

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  9. Chikatoshi Kai

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  10. Matthias Harbers

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  11. Yoshihide Hayashizaki

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  12. Piero Carninci

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Corresponding authors

Correspondence toMatthias Harbers,Yoshihide Hayashizaki orPiero Carninci.

Supplementary information

Supplementary Fig. 1

Flowchart for the high-throughput preparation of full-length enriched cDNAs. (PDF 149 kb)

Supplementary Table 1

Oligonucleotides for use in CAGE protocol. (DOC 26 kb)

Supplementary Table 2

CAGE Linker Oligos. (PDF 312 kb)

Supplementary Table 3

HPLC gradient used for fractionation of concatemers. (DOC 563 kb)

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

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