Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines
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We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of >298 high-quality SAGE libraries and >30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection. Several RNA isolation methods were used to handle the diversity of samples processed, and various measures were applied to minimize ditag PCR carryover contamination. Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies. Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure, linker or cross-species ditag contamination, and yield of high-quality tags per sequence read. Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy. From the libraries generated, we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of ∼5 million tags. Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts. Rather than the usual practice of discarding such tags, we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data, which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling.
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The longSAGE protocol generates tags that are 21 bp long and provides enhanced efficiency and accuracy of tag-to-gene mapping.
“SAGE has been improved by the development of a longSAGE protocol, which generates tags that are 21 bp long and provides enhanced efficiency and accuracy of tag-to-gene mapping [30-32].”
From A systematic screen for genes expressed in definitive endoderm by Serial Analysis of Gene Expression (SAGE) (2007) · cites this paper as [32]
The Results and Discussion identify LongSAGE tags as 21 bp, and the introduction says longer tags improve the specificity of tag-to-gene mapping. The paper reports improved cloning and sequencing efficiency, but the cited passages do not establish improved efficiency of tag-to-gene mapping itself. Page numbers were not available in our copy. (LLM, conf=0.84)
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Cites
The RIN: an RNA integrity number for assigning integrity values to RNA measurements
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A Global Map of p53 Transcription-Factor Binding Sites in the Human Genome
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Using the transcriptome to annotate the genome
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Flagged first, then by citations
Research works printed but not linked: 14
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Sorted by citations
Specific expression of long noncoding RNAs in the mouse brain
Mercer, T. R., Dinger, M. E., Sunkin, S. M., Mehler, M. F., & Mattick, J. S. (2008) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0706729105 · Cited by 1,103 · see citing works
Control of embryonic stem cell lineage commitment by core promoter factor, TAF3
Liu, Z., Scannell, D. R., Eisen, M. B., & Tjian, R. (2011) · Cell · doi:10.1016/j.cell.2011.08.005 · Cited by 167 · see citing works
Hou, J., Charters, A. M., Lee, S. C., Zhao, Y., Wu, M. K., Jones, S. J., Marra, M. A., & Hoodless, P. A. (2007) · BMC Developmental Biology · doi:10.1186/1471-213x-7-92 · Cited by 56 · see citing works
“Conclusion: The high efficiency of this endoderm screen indicates that our approach can be successfully used to analyze and validate the vast amount of data obtained by the Mouse Atlas of Gene Expression Project.”
BackgroundMethodInfluentialSelf-citation
- Authors
- Khattra, J., Delaney, A. D., Zhao, Y., Siddiqui, A., Asano, J., McDonald, H., Pandoh, P., Dhalla, N., Prabhu, A.-L., Ma, K., Lee, S., Ally, A., Tam, A., Sa, D., Rogers, S., Charest, D., Stott, J., Zuyderduyn, S., Varhol, R., Eaves, C., Jones, S., Holt, R., Hirst, M., Hoodless, P. A., & Marra, M. A.
- Affiliations
- BC Cancer Agency (CA)
- Corresponding author
- Marra, Marco A.
- ORCID
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2006-11-29
- Volume
- 17
- Issue
- 1
- Pages
- 108-116
- ISSN
- 1088-9051 (ISSN-L)
- PMC
- PMC1716260
- OpenAlex
- W2161797935
- Cited by
- 40
- References
- 4
- Citations by year
- 2023: 1 · 2015: 1 · 2014: 1 · 2013: 1 · 2012: 4
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genome Research (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Genome Canada
- Canada's Michael Smith Genome Sciences Centre
- Michael Smith Health Research BC
- Canadian Institutes of Health Research
- National Cancer Institute
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Keywords
- serial analysis of gene expression, cell lines, RNA extraction, gene expression profiling, sequencing quality assessment
- MeSH terms
- Animals, Cell Line, Cell Separation, Flow Cytometry, Humans, Software, Zebrafish, Gene Library, Genome, Caenorhabditis elegans, Sequence Analysis, DNA, Gene Expression Profiling, Databases, Nucleic Acid, Microdissection, Mice, Embryonic Stem Cells
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