title: 454 sequencing of gonad transcriptome library
design: Isolation of total RNA was done with the RNeasy Midi Kit (Qiagen), using 70 – 100 mg tissue (without addition of ß-mercaptoethanol during the RNA cleanup step).  Poly-A  RNA was isolated with the Oligotex mRNA mini kit (Qiagen). First strand cDNA synthesis was performed with RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) where the synthesis time was reduced from 1 hour to 7.5 minutes to obtain cDNA of the desired length for 454 library preparation. Second strand synthesis was performed with DNA polymerase I, E.coli (Fermentas) according to standard protocol. The double stranded cDNA solutions were cleaned up with MinElute PCR Purification Kit (Qiagen). Libraries for 454 sequencing were produced and sequenced following the Roche protocol for shotgun Titanium standard library preparation.