Transcriptome of 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]. Six wild specimens (three male and three female) 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_west population