title: Eustoma exaltatum subsp. russellianum Transcriptome or Gene expression
design: Total RNA was extracted according to a CTAB protocol. Three-gram samples were ground into powder in liquid nitrogen, mixed with 30mlof 2XCTAB solution (1.4M NaCl, 0.1M Tris-HCl pH9.5, 20mM EDTA, 2% CTAB, and 1% 2-mercaptoethanol),and incubated at 65ºC for 10 min.The solution was centrifuged at 5,000rpm for 10min at room temperature. The supernatant was collected and extracted with a 25:25:1 mixture of phenol/chloroform/isoamyl alcohol (PCI). RNA was precipitated by the addition of propanol. The precipitate was resuspendedin 2.0mlof water and precipitated with 2 M LiCl, followed by a series of PCI and chloroform extractions. Finally, the sample was collected by ethanol precipitationand resolved in 50µlof water.  The total RNA extracted from each sample was combined, and the cDNA sequences of the samples were determined by Operon Biotechnologies (Tokyo, Japan) as described below. cDNA was synthesised using random primers, and a normalised library consisting of fragmented cDNA was then constructed. Normalisation was performed by one cycle of denaturation and reassociation of the cDNA. Reassociated ds-cDNA was separated from the remaining ss-cDNA bypassing the mixture over a hydroxyapatite column. After hydroxyapatite chromatography, the ss-cDNA was amplified through 11 PCR cycles.Fragments of cDNA with a length of 500-700bp were sequenced using a Roche Genome Sequencer FLX System.