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454 Transcriptome Sequencing

Plant Material and RNA Extraction

Three European families of Silene vulgaris were chosen to represent the major axes of genetic differentiation found in this species. Plants from each of these families were grown up in the greenhouse. In order to maximize gene identification, we extracted total cellular RNA from multiple tissue types (leaf, flower, and root). RNA samples from multiple families and multiple tissue types were pooled (without tagging) for a single library construction.

cDNA Library Construction and 454 Sequencing

The pooled RNA sample was submitted to the Indiana University Center for Genomics and Bioinformatics (CGB), which produced a normalized, polyA-selected cDNA library. This library was sequenced on a 454 GS FLX with titanium reagents by the University of Virginia Genomics Core Facility. A single full 454 plate was run, producing a total of 380 Mb of sequence data with an average read length of 392 bp (prior to removal of custom library adaptors).