BMC Genomics Methodology article A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA Claytus Davis*, Zeev Barvish and Inna Gitelman BioMed Central Open Access Address: Department of Virology and Developmental Genetics, Faculty of Health Science, Ben Gurion University of the Negev, Beer Sheva, Israel Email: Claytus Davis* - clay@bgu.ac.il; Zeev Barvish - barvish@bgu.ac.il; Inna Gitelman - gitelman@bgu.ac.il * Corresponding author Published: 9 October 2007 BMC Genomics 2007, 8:363 doi:10.1186/1471-2164-8-363 This article is available from: http://www.biomedcentral.com/1471-2164/8/363 Received: 29 December 2006 Accepted: 9 October 2007 © 2007 Davis et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. Results: We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed) total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. Conclusion: We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired. Background Almost the entire trans-acting fraction of genetic informa- tion is represented by the transcriptome, the population of transcribed sequences in a cell. In terms of complexity, much of the functional transcriptome of eukaryotic cells has traditionally been considered poly-adenylated and translated. In terms of quantity, this poly-adenylated frac- tion constitutes only 3–6% of the total RNA population. For these reasons, experimental representation of eukary- otic transcriptomes was usually done by constructing cDNA libraries from the poly A+ fraction of the RNA pop- ulation. All such libraries do not, by design, represent the entire trans-acting genetic information. They lack repre- sentation of non-coding but functional RNAs (ncRNA), Page 1 of 7 (page number not for citation purposes)