BMC Genomics 2007, 8:363 http://www.biomedcentral.com/1471-2164/8/363 the added advantage of reducing the formation of intramolecular secondary structure that can interfere with priming and reducing the expected bias for the 5' ends of full-length molecules. (3) The quality of the cDNA syntheses and subsequent PCR amplification steps was evaluated by tracing the reac- tions with 32P-dCTP and then determining the incorpo- rated fraction to estimate the yield and agarose gel electrophoresis to gauge the quality of the reaction. Oligonucleotide quality was found to be important. We used HPLC grade oligonucleotides for the library con- struction and self-subtraction. DRP1 is 5'P-GCTCGCCCT CGCGGCGCGCCNNNNNT. The lone-linker LL1 is the annealed product of LL1F and LL1R 5'CTGGCTCGCCCTCGCGGATCCG (LL1F) AGACCGAGCGGGAGCGCCTAGGC 5' (LL1R) (4) LL1P is 5'CTGGCTCGCCCTCGCGG. The correct amount of template and the number of cycles were deter- mined empirically. Using the reaction conditions described, a yield of between 1 and 5 µg was typical. A yield greater than this may be stressing the reaction, result- ing in partial reaction products and other artifacts. (5) The hydroxylapatite was de-fined prior to use by resus- pending the powder in a large volume of annealing buffer, allowing the matrix to settle by gravity, and removing the still slightly cloudy upper phase. This was repeated 3–5 times. 1 ml of hydrated hydroxylapatite binds approxi- mately 100 µg of DNA. (6) Failure to completely remove all hydroxylapatite results in binding of ssDNA as the phosphate concentra- tion drops during the next buffer exchange step. (7) Depending upon the extent of annealing, the concen- tration of remaining ssDNA may be low enough to result in a significant fraction binding to the filter membrane. To prevent this, the centricons should be passivated in 5% Tween-20 for one hour followed by four rinses with ddH2O. Authors' contributions CD made the libraries and assisted in writing the paper. ZB assisted in making the libraries and completed the analysis. CD and ZB contributed equally to this work. IG participated in the design of the study, analysis of results, and writing the paper. Additional material Additional file 1 The supplementary data file lists properties of cDNA sequences that match genomic loci in the sequence databases. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-8-363-S1.pdf] Acknowledgements This work was supported by an Israel Science foundation grant to CD and a Binational USA/Israel Science Foundation grant to IG. We thank Dr. Linda Holland for the embryonic Amphioxus sample. References 1. Nakamura A, Amikura R, Mukai M, Kobayashi S, Lasko PF: Require- ment for a noncoding RNA in Drosophila polar granules for germ cell establishment. Science 1996, 274(5295):2075-2079. 2. 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