Total RNA was extracted using Tri-Reagent (Sigma-Aldrich, USA) from five main tissues: mantle, gills, digestive gland and abductor muscle. RNA from all individuals (n = 10) and all five tissues were polled in equal quantities (800 ng) and enriched for mRNA using GenElute mRNA miniprep Kit (Sigma-Aldrich, St. Louis, Missouri, USA). The final enriched high-quality mRNA was used for cDNA synthesis.
cDNA was synthetized using MINT Kit (Evrogen, Moscow, Russia), following the manufacturer’s instructions, with a modified oligo-dT (5′-AAGCAGTGGTATCAACGCAGAGTCGCAGTCGGTACTTTTTTCTTTTTTV-3′) which has a poly-T stretch broken by the inclusion of an internal C to minimize the potential for 454 sequencing problems in this homopolymer stretch [40] (link).
The cDNA library was normalized using the Trimmer Direct kit (Evrogen, Moscow, Russia) to prevent over-representation of the most common transcripts. Finally, approximately 10 µg of cDNA were used to construct two cDNA GS Junior 454 libraries that were sequenced separately. Roche GS Junior 454 pyrosequencing runs were conducted at the Biology Institute at the Federal University of Rio de Janeiro, Brazil.
cDNA was synthetized using MINT Kit (Evrogen, Moscow, Russia), following the manufacturer’s instructions, with a modified oligo-dT (5′-AAGCAGTGGTATCAACGCAGAGTCGCAGTCGGTACTTTTTTCTTTTTTV-3′) which has a poly-T stretch broken by the inclusion of an internal C to minimize the potential for 454 sequencing problems in this homopolymer stretch [40] (link).
The cDNA library was normalized using the Trimmer Direct kit (Evrogen, Moscow, Russia) to prevent over-representation of the most common transcripts. Finally, approximately 10 µg of cDNA were used to construct two cDNA GS Junior 454 libraries that were sequenced separately. Roche GS Junior 454 pyrosequencing runs were conducted at the Biology Institute at the Federal University of Rio de Janeiro, Brazil.
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