nanoCAGE/CAGEscan Library

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nanoCAGE Library

nanoCAGE/CAGEscan is a modified technique of CAGE that enables genome-wide promoter analysis of small-amount of samples. Utilizing a template-switching reaction and a semi-suppressive PCR, amount of sample required is significantly reduced. CAGEscan basically sequence and map short tags derived from the 3’-end of cDNA (5’-end of mRNA), thereby quantifying the frequency of tag sequences. Like CAGE, nanoCAGE/CAGEscan can accurately identify promoter sites for each transcript and obtain expression profile of respective promoters in genome-wide fashion. Several articles performing transcriptome analysis with nanoCAGE in human and mouse have been published.

With a sensitivity one thousand times higher than CAGE, nanoCAGE/CAGEscan presents powerful new possibilities for the analysis of samples with an amount as little as 50 ng of total RNA. NanoCAGE/CAGEscan overcomes the difficulty on obtaining large quantity of cells for canonical CAGE analysis, supplying a possibility for diagnosis of diseases such as detection of cancer and neuron disorder in future.

As the joint developer with RIKEN, DNAFORM library preparation service provides the end-to-end solution for gemone-wide semi-quantitative expression analysis including library construction, high- throughput sequencing with the Illumina HiSeq platforms and data analysis.

Applications

  • Genome-wide gene expression analysis with tiny samples
  • Prediction of promoter sites with small amount of sample

How to choose CAGE and nanoCAGE/CAGEscan

Library Advantage Disadvantage
CAGE Quantitative expression profile without PCR bias 5 μg of total RNA is reqired
nanoCAGE/CAGEscan Only 50 ng of total RNA is required.
3'-end analysis with the same library is possible
PCR step during the library preparation may cause PCR biases on the expression profile.

nanoCAGE/CAGEscan Library Service

Technical details

Item Specification Comment
Total RNA required 50 ng / 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
Number of reads per channel guaranteed 25 M reads/lane
Approx. 40 – 50 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 50% of tags map to unique mapping position 1 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.
  • Estimated Turn-around-times: About 2 months
  • Documentation: Report on nanoCAGE/CAGEscan library preparation in a flat text file format.

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Technical Information

How to sequence nanoCAGE library

RIKEN has applied for the pooled sequencing nanoCAGE libraries (multiple barcodes) the following conditions: They used DNA solutions adjusted to 15 pM of DNA for loading on an Illumina HiSeq2500. The molarity was calculated by ONLY taking into account the molecules in the 100–1000 bp size range. When using only a single barcode, problems may occur with the Illumina basecaller, and therefore RIKEN used 10 pM DNA solutions.

Note if you are using qPCR to determine the DNA concentration for loading onto an Illumina sequencer, the experiment will provide the concentration of ALL DNA fragments including the long fragments that will not yield sequencing reads on an Illumina platform. You will have to adjust the molarity for loading considering the amount of DNA fragments in the 100-1000 bp range. The Bioanalyzer has a function to calculate the molarity on size ranges in its electrophoregram section.

Please refer to the following site for cluster generation.
http://fantom.gsc.riken.jp/5/sstar/OP-HiSeq2000-sequencing-cBot-v3.0

References

nanoCAGE/CAGEscan

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  2. M. Salimullah et al, NanoCAGE: a high-resolution technique to discover and interrogate cell transcriptomes, Cold Spring Harb Protoc. 2011 (2011)
  3. D. Tang et al, Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching, Nucleic Acids Res, 41, e44 (2013)
  4. M. Harbers et al, Comparison of RNA- or LNA-hybrid oligonucleotides in template-switching reactions for high-speed sequencing library preparation, BMC Genomics, 14, 665 (2013)
  5. CH. Chien, Identifying transcriptional start sites of human microRNAs based on high-throughput sequencing data, Nucleic Acids Res, 39, 9345-956 (2011)
  6. C. Plessy et al, Promoter architecture of mouse olfactory receptor genes, Genome Res, 7, 528-534 (2011)

Others

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