Transcriptome characterization of the South African abalone Haliotis midae using pyrosequencing
Specimens were collected across the geographical range of H. midae: west coast [Saldanha bay]; south coast [Witsand] and east coast [Riet Point]. Cultured specimens (F1) were collected from two aquaculture facilities. Six cultured specimens (three female and three male) were selected from each of the three geographic regions. Tissue was collected from five organs from each individual: muscle, ganglion, hepato-pancreas, gonad and gill.Biopsied tissues were immediately placed in RNALater® (Ambion®) solution and stored at -20°C until RNA extraction could be performed. RNA was extracted from 1-2 g of tissue from each organ and individual, separately. Denaturing agarose gel electrophoresis [2% agarose; 1X 3-(N-morpholino) propanesulfonic acid (MOPS) buffer] was performed to determine RNA integrity and absorbance measurements were taken using the NanoDrop® ND-1000 spectrophotometer to assess RNA purity and concentration. The SuperScript® Double-Stranded cDNA Synthesis Kit (Invitrogen) was used according to the manufacturer’s specifications to generate double stranded cDNA. Oligo (dT) primers were used to selectively amplify poly-adenylated mRNA. Molar amounts to provide a final pooled sample for each population group containing 2 μg of cDNA at a concentration of 100 ng/μl. Pooled cDNA was used for pyrosequencing, according to the manufacturer’s protocol. Pyrosequencing was conducted on a full-plate of the 454 GS FLX platform (Roche).
Stellenbosch University
H.midae sequencing project