Roche 454 of a pool of Pecten maximus tissues
RNA isolation was carried out using Trizol (Invitrogen). Purification of RNA after DNase I treatment was performed with RNeasy minikit (Qiagen). Concentration and purity of RNA was calculated with a NanoDrop ND1000 spectrophotometer and the quality was checked with a Bioanalyzer 2010 (Agilent Technologies). A full-length-enriched double stranded cDNA synthesis was performed 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. The method involves denaturation-reassociation of cDNA, followed by a digestion with a Duplex-Specific Nuclease (DSN) enzyme. The enzymatic degradation occurs primarily on the highly abundant cDNA fraction. The single-stranded cDNA fraction was then amplified twice by sequential PCR reactions according to the manufacturer's protocol. Normalized cDNA was purified using the QIAquick PCR Purification Kit (Qiagen USA, Valencia, CA).   500 ng of normalized cDNA were used to generate a 454 library. cDNA was fractionated into small, 300- to 800-basepair fragments and the specific A and B adaptors were ligated to both the 3' and 5' ends of the fragments. The A and B adaptors were used for purification, amplification, and sequencing steps. Sequencing runs were performed on the GS-FLX using Titanium chemistry. All reagents and protocols used were from Roche 454 Life Sciences, USA. RNA was normalized, processed and sequenced by the Unitat de Genòmica (CCiT-UB, Barcelona, Spain)