title: 20081212-Tragopogon-dubius-FLX 2674-4 (3911)
design: Seeds were germinated and grown in an air-conditioned greenhouse with supplementary lighting at the University of Florida (Gainesville, FL,USA). RNA was extracted from leaf tissue of T. dubius 2674-4 (ID no. 3911). Basal leaf tissue from each plant was flash frozen and ground in liquid nitrogen using a pestle and mortar. RNA extractions were performed following a portion of the CTAB DNA extraction protocol (Doyle & Doyle 1987) and subsequent use of the RNeasy Plant Mini Kit (Qiagen, Stanford, CA, USA) with on-column DNase digestion. This was followed by an RNA cleanup using the protocol of the RNeasy Plant Mini Kit. These extractions were quality-checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). A normalized cDNA library 201 was produced via the following method. The Evrogen MINT cDNA synthesis kit (Evrogen, Moscow, Russia) was used to produce double-stranded cDNA following the manufacturer’s protocol. This cDNA was cleaned using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). The Evrogen TRIMMER cDNA normalization kit (Evrogen, Moscow, Russia) was used to normalize and amplify the cDNA library, following the manufacturer’s instructions. In the normalization step, a 0.5 dilution of the duplex-specific nuclease (DSN) was found to be optimal. In the amplification step, 12 cycles were found to be optimal. The resulting normalized library was used for 454 sequencing. 454 sequencing was performed as described in the supplementary material and methods to Margulies et al. (2005) with slight modifications as specified by 454 Life Sciences. Briefly, cDNA was sheared by nebulization to a size range of 300 to 800 bp. DNA fragment ends were repaired and phosphorylated using T4 DNA polymerase and T4 polynucleotide kinase. Adaptor oligonucleotides “A” and “B” supplied with the 454 Life Sciences sequencing reagent kit were ligated to the DNA fragments using T4 DNA ligase. Purified DNA fragments were hybridized to DNA capture beads and clonally amplified by emulsion PCR (emPCR). DNA capture beads containing amplified DNA were deposited on a 70 x 75 mm PicoTiter plate, and DNA sequences determined using the GS-FLX instrument. This resulted in 822,594 EST sequences.