EST characterization and polymorphism detection of two Artemisia tridentata subspecies
The cDNA used for 454 titanium pyrosequencing was synthesized using 1µg of total RNA using the SMART cDNA synthesis kit (Clontech Laboratories, Inc., Mountain View, CA) but the cDNA synthesis primer for first strand synthesis was replaced by a modified oligo-dT primer (5’-AAGCAGTGGTATCAACGCAGAGTCGCAGTCGGTACTTTTTTCTTTTTTV-3’). To reduce potential skewness in transcriptional representation, the cDNA library was normalized using the Trimmer Kit (Evrogen, Moscow, Russia). The poly-T stretch in the primer is broken by inserting an extra Cytosine to minimize the potential sequencing problems due to the presence of a long ploy-A homopolymer stretch. A revised version of the Eli Meyer (2009) protocol was used for the cDNA library preparation. The cDNA library was further processed for ligating adaptors and barcodes and single strand selection using the GS FLX Titanium General Library Preparation Kit (Roche Applied Sciences, Basel, Switzerland) with modifications. About 1 µg cDNA of each subspecies was used for a quarter-plate sequencing run using 454 GS-FLX pyrosequencing technology. The full revised protocol used for cDNA library preparation is made available with the Supplemental Materials. The sequencing was done at the Brigham Young University DNA sequencing center.