Abstract
Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.
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Acknowledgments
This work was supported by a Research Grant for the RIKEN Genome Exploration Research Project to Y.H and a grant of the Genome Network Project, both from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.
We would like to thank Dr. Carninci P. for his advice, and also the colleagues in the Genome Exploration Research Group at the Genomic Sciences Center (GSC), RIKEN Yokohama Institute, for all the support.
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Harada, M., Hayashizaki, Y. (2009). Preparation of Full-Length cDNA Libraries: Focus on Metazoans. In: Parkinson, J. (eds) Expressed Sequence Tags (ESTs). Methods in Molecular Biology, vol 533. Humana Press. https://doi.org/10.1007/978-1-60327-136-3_5
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DOI: https://doi.org/10.1007/978-1-60327-136-3_5
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