pyrosequencing of the ovary and testis from wild and domesticated black tiger shrimps Penaeus monodon: Sample OVD
Broodstock were both live-caught from the Andaman Sea and grow-out ponds with with gonadosomatic index (GSI) values of 5.37 amd 0.72, respectively. Ovaries and testes of P. monodon broodstock were dissected and ground in liquid nitrogen. Total RNA was extracted from the tissue using TRI-Reagent according to manufacturer’s instruction (Molecular Research Center, USA). Contaminated genomic DNA was removed by treatment with DNase I (0.15 U/µg total RNA) at 37°C for 30 min.  The quality and quantity of the RNA were assessed on an agarose gel electrophoresis and NanoDrop (ND-8000). Poly-A mRNA sample (200 ng) was isolated using Absolutely mRNA Purification Kit (Agilent Technologies) and fragmented in 10x fragmentation buffer (0.1 M ZnCl2, 0.1 M Tris-HCl, pH7.0) at 70°C for 30 sec.  The reaction was stopped by adding 2 µl 0.5 M EDTA and 28 µl 10 mM Tris HCl, pH7.5.  The mRNA sample was cleaned using Agencourt RNAClean reagent (Beckman Coulter), washed with 200 µl 70% EtOH, air dried and eluted in 20 µl 10 mM Tris HCl, pH7.5.  Fragmented mRNA samples were converted to double-stranded cDNA with the cDNA Synthesis System Kit (Roche Applied Science) using random primers and AMV Reverse Transcriptase.  A cDNA library for 454 pyrosequencing was prepared according to the cDNA Rapid Library Preparation protocol (Roche Applied Sciences).  The cDNA library was amplified in emulsion PCR and subject to pyrosequencing on half of a picotitre plate on the Genome Sequencer (GS) FLX Titanium platform (Roche Applied Sciences).