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The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects, are often difficult to provide for further studies or third parties due to the consumptive nature of the samples. Although now widely distributed over the Pacific coasts of Asia, Australia and North America, individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries. In this article, we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries. These libraries were then compared to the publicly available oyster genome, and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I (COX1) and NADH Dehydrogenase (ND). The suitability of the generated cDNA library is also demonstrated by cloning and expression of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS), which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.

Large-scale RNA interference (RNAi) screens in mammalian cells have mainly used synthetic small interfering RNA (siRNA) or short hairpin RNA (shRNA) libraries. The RNAi triggers for both of these approaches were designed with algorithm-based predictions to identify single sequences for mRNA knockdown. Alternatives to these approaches have recently been developed using enzymatic methods. Here we describe the concepts of enzymatically prepared shRNA and siRNA libraries, and discuss their strengths and limitations.

We present an analysis of the chicken (Gallus gallus) transcriptome based on the full insert sequences for 19,626 cDNAs, combined with 485,337 EST sequences. The cDNA data set has been functionally annotated and describes a minimum of 11,929 chicken coding genes, including the sequence for 2260 full-length cDNAs together with a collection of noncoding (nc) cDNAs that have been stringently filtered to remove untranslated regions of coding mRNAs. The combined collection of cDNAs and ESTs describe 62,546 clustered transcripts and provide transcriptional evidence for a total of 18,989 chicken genes, including 88% of the annotated Ensembl gene set. Analysis of the ncRNAs reveals a set that is highly conserved in chickens and mammals, including sequences for 14 pri-miRNAs encoding 23 different miRNAs. The data sets described here provide a transcriptome toolkit linked to physical clones for bioinformaticians and experimental biologists who wish to use chicken systems as a low-cost, accessible alternative to mammals for the analysis of vertebrate development, immunology, and cell biology.

RNA interference (RNAi) induced by small interfering (siRNA) or short hairpin RNA (shRNA) is an important research approach in mammalian genetics. Here we describe a technology called enzymatic production of RNAi library (EPRIL) by which cDNAs are converted by a sequence of enzymatic treatments into an RNAi library consisting of a vast array of different shRNA expression constructs. We applied EPRIL to a single cDNA source and prepared an RNAi library consisting of shRNA constructs with various RNAi efficiencies. High-throughput screening allowed us to rapidly identify the best shRNA constructs from the library. We also describe a new selection scheme using the thymidine kinase gene for obtaining efficient shRNA constructs. Furthermore, we show that EPRIL can be applied to constructing an RNAi library from a cDNA library, providing a basis for future whole-genome phenotypic screening of genes.