title: Lake Alexandrina - Asexual - 454 GS FLX Titanium cDNA sequencing
design: This sample is the 454-sequencing of cDNA from five Potamopyrgus antipodarum adults taken from Lake Alexandrina, New Zealand. Total RNA was extracted from whole snail bodies (pooled within lineages prior to extraction) using TRIzol reagent per the manufacturer’s instructions. RNA was then quantified on a ND-1000 Spectrophotometer (Thermo Fisher Scientific, Inc.; Wilmington, DE). cDNA synthesis and normalization were carried out using the Evrogen Trimmer-Direct cDNA normalization kit per the manufacturer’s instructions. We used 5 µg of the obtained normalized cDNA obtained from each lineage extraction for 454 sequencing. Sample fragmentation, single stranded library preparation, and emPCR were carried out according to the manufacturer’s protocol.  The clonally amplified cDNA attached to sequencing beads was then loaded on a four-chambered PicoTiterPlate and sequenced using the Titanium sequencing chemistry.