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 ×
Gene Expression Quantification 1
Determining Promoter Site × ×
Motif Finding for Transcription Factor Binding Site × 2 × 2
Identification of Bidirectional Enhancer RNA × × ×
Determining Transcription Start/1st Exon Site × ×
Determining Gene Structure (intron/exon, alternative splicing variants) × 3 × ×
Duration of Work Process 3
Library Preparation Complicatedness × 4
Data Analysis Tools
  • 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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  3. Yu NY et al, Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium, Nucleic Acids Res, 43(14), 6787-6798 (2015)
  4. and more
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