CAGE - What can it do for you?

  • Highly sensitive and accurate quantitative transcriptome analysis not only as a gene, but as a Transcription Start Site (TSS).
  • Reliable way to discover Transcription Factor binding motif based on the true TSS nearby the actual promoter.
  • Unique and powerful tool to discover alternative promoters for all endogenous genes.
  • Help you to discover new biomarker and/or bidirectional enhancer RNA.
  • Clarifying the heterogeneity and complexity of transcriptomes from the view of Cause of Transcription instead of expression profiling.
  • Linking expression values to promoter sites for a better understanding of how signaling pathways regulate transcription.
  • PCR-free operation procedures provide unbiased quantification of transcript amount.

Difference from RNA-Seq and other gene expression analysis techniques

Different from Microarray and RNA-seq, CAGE is able to accurately identify transcriptional start sites (TSSs) and the corresponding promoter regions through sequencing the 3’ end of cDNA (5’ end of RNA). This makes CAGE a powerful tool to analyze the gene regulation in the TSSs level, enabling analysis of the gene regulated by multiple alternative promoters. Therefore, CAGE can serve as a new perspective approach for genome annotation, by elucidating transcriptional signaling cascades, and performing other functions.

Comparison among major gene expression analysis techniques
CAGE RNA-seq SAGE Microarray
de novo Gene Finding good good good N/A
Gene Expression Quantification superior 1
※ free of PCR bias unaffected by gene size
good good average
Determining Promoter Site superior average N/A N/A
Motif Finding for Transcription Factor Binding Site superior average average 2
※ depending on known 5' end sequence information
average 2
※ depending on known 5' end sequence information
Identification of Bidirectional Enhancer RNA superior N/A N/A N/A
Determining Transcription Start/1st Exon Site superior average N/A N/A
Determining Gene Structure (intron/exon, alternative splicing variants) N/A average3
※ depending on sequence depth
N/A N/A
Duration of Work Process average average average good
Library Preparation Complicatedness ※Long Time 4 (8 days) average average easy
Data Analysis Tools average good average good

("N/A" means not applicable)

  • 1 free of PCR bias and unaffected by gene size
  • 2 depending on known 5' end sequence information
  • 3 depending on sequence depth
  • 4 8 days

What is the CAGE method?

Cap Analysis of Gene Expression (CAGE) is a method for promoter identification and transcription profiling developed by RIKEN (Patent Number: US 6174669, US 6221599, US8809518, etc.). CAGE utilizes a “cap-trapping” technology based on the biotinylation of the 7-methylguanosine cap of Pol II transcripts, to pull down the 5’-complete cDNAs reversely transcribed from the captured transcripts. Through a massive parallel sequencing of the 5’ end of cDNA and analysis of the sequenced tags, transcription start sites and transcripts amount are inferred on a genome-wide scale. Thus, CAGE provides an effective genome-wide transcriptional profiling as an alternative to microarray and RNA-seq.

Item Specification Comment
Total RNA required 5 μg / sample Total RNA preferable
RNA entry QC Bioanalyzer We perform entry QC on all samples
DNA amount of CAGE library Several ng DNA fragments ready for illumine NGS sequencer
Sequencing platform Illumina HiSeq 2000/2500
Number of reads per lane guaranteed 75 M reads/lane
Approx. 100 - 150 M reads / lane in average
Standard conditions: 8 - 12 samples per lane.
Optional extra sequencing Number of lanes per analysis Additional lanes are available with additional charge.
Mapping rate About 75% of tags map to unique mapping position 4 M mappable CAGE tags / sample is guaranteed.
Sequence data Provided with Illumina file format Delimited text files holding sequence information and quality scores.
Data Analysis Mapping positions Tables/flat files: number of raw reads, number of extracted tags, number of mapped tags, etc.

Sample Data Download

Downlod the raw fastq data, processed fastq data, reference genome, and analysis results
* The file may take some time to download as the total data size is 32.6GB.
Download

How does CAGE work?

First strand cDNA synthesis is performed using our proprietarily owned technology facilitating thermostable reverse transcription and random priming. Random Priming allows for the finding of non-polyadenylated RNA as well. RNAs without the 5’-cap such as rRNA, truncated RNA, and incompletely reversely transcribed RNAs are eliminated by cap-trapping.

After selection of full-length cDNA, adaptors are ligated to the 5'-ends of full-length enriched cDNAs as well as the 3'-ends in the strand-specific manner. CAGE Tags are ready for high-throughput sequencing after the 2nd strand synthesis.

CAGE tags are mapped to the reference genome to identify TSS and their related promoter regions. In addition, comparison of mapping positions to defined transcripts provides an annotation of the CAGE tag. The number of CAGE tags mapped at specific locations in the genome provides quantification on promoter activities on a genome-wide scale.

Hence, CAGE can contribute to projects in Genome Annotation, Gene Discovery, Gene Expression Profiling, and Promoter Identification. The Reference List showed examples on how CAGE has been successfully used in studies related to transcriptome analysis and promoter identification. Our CAGE protocols are based on a well-established procedure capable of obtaining a large number of tags.

Why we can trust the CAGE analysis?

Extensively used within the

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Shipping and Packing

No charge for shipping within Japan.
Shipping outside Japan, extra fee will be charged. Please refer our Shipping, Packing Charge Information

Shipping and Packing Charge
The library will be shipped at RT as dried pellet.

References

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  2. Tsuru A et al, Nonpathogenic Cutibacterium acnes Confers Host Resistance against Staphylococcus aureus, Microbiol Spectr, 9(2), e0056221
  3. Takata N et al, Prior secondary cell wall formation is required for gelatinous layer deposition and posture control in gravi‐stimulated aspen, Plant J, 108(3), 725-736
  4. and more
  5. Okamura Y et al, A defective interleukin-17 receptor A1 causes weight loss and intestinal metabolism-related gene downregulation in Japanese medaka, Oryzias latipes, Sci Rep, 11(1), 12099
  6. Yano K et al, PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA, J Biol Chem, 297(1), 100882
  7. Takeuchi S et al, Small extracellular vesicles derived from interferon-γ pre-conditioned mesenchymal stromal cells effectively treat liver fibrosis, NPJ Regen Med, 6(1), 19
  8. Watanabe M et al, Esm1 and Stc1 as Angiogenic Factors Responsible for Protective Actions of Adipose-Derived Stem Cell Sheets on Chronic Heart Failure After Rat Myocardial Infarction, Circ J, 85(5), 657-666
  9. Bamba M et al, Massive rhizobial genomic variation associated with partner quality in Lotus–Mesorhizobium symbiosis, FEMS Microbiol Ecol, 96(12), fiaa202
  10. Murakami K et al, A genome-scale CRISPR screen reveals factors regulating Wnt-dependent renewal of mouse gastric epithelial cells, Proc Natl Acad Sci U S A, 118(4), e2016806118
  11. Gacita A et al, Altered Enhancer and Promoter Usage Leads to Differential Gene Expression in the Normal and Failed Human Heart, Circ Heart Fail, 13(10), e006926
  12. Naka K et al, The lysophospholipase D enzyme Gdpd3 is required to maintain chronic myelogenous leukaemia stem cells, Nat Commun, 11(1), 4681
  13. Mae S et al, Expansion of Human iPSC-Derived Ureteric Bud Organoids with Repeated Branching Potential, Cell Rep, 32(4), 107963
  14. Okuno T et al, Zygotic Nuclear F-Actin Safeguards Embryonic Development, Cell Rep, 31(13), 107824
  15. Ito Y et al, Roles of Elm1 in antifungal susceptibility and virulence in Candida glabrata, Sci Rep, 10(1), 9789
  16. Sakamoto S et al, Id genes are required for morphogenesis and cellular patterning in the developing mammalian cochlea, Dev Biol, 460(2), 164-175
  17. Ueno D et al, Different Plant Species Have Common Sequence Features Related to mRNA Degradation Intermediates, Plant Cell Physiol, 61(1), 53-63
  18. Higuchi C et al, Perturbation of maternal PIASy abundance disrupts zygotic genome activation and embryonic development via SUMOylation pathway, Biol Open, 8(10), bio.048652
  19. Kato M et al, Clostridium butyricum MIYAIRI 588 Increases the Lifespan and Multiple-Stress Resistance of Caenorhabditis elegans, Nutrients, 10(12), 1921