title: Reproductive turbot tissue 454 Titanium sequencing project
design: Full-length-enriched double stranded cDNA was synthesized from 1,5 µg of pooled total RNA using MINT cDNA synthesis kit (Evrogen, Moscow, Russia) according to manufacturer's protocol, and was subsequently purified using the QIAquick PCR Purification Kit (Qiagen USA, Valencia, CA). The amplified cDNA was normalized using Trimmer kit (Evrogen, Moscow, Russia) to minimize differences in representation of transcripts(Shagin et al. 2002; Zhulidov et al. 2004). The single-stranded cDNA fraction was then amplified twice by sequential PCR reactions according to manufacturer's protocol. Normalized cDNA was purified using the QIAquick PCR Purification Kit (Qiagen USA, Valencia, CA). 5 µg of normalized cDNA were used to generate a 454 library. cDNA was fractionated into small, 300- to 800-base pair fragments and the specific A and B adaptors were ligated to both the 3' and 5' ends of the fragments and used for purification, amplification, and sequencing steps.