parents treated with Diuron 2
RNASeq library construction and sequencing were provided by Acobiom Company (Montpellier). Two libraries per oyster group (SWC, SC and D) were constructed as biological duplicates. Total RNA from 10 spats was pooled in equal quantities to reach 5 µg as template for each cDNA library. The Illumina mRNA-Seq singled-end was used according to the manufacturer's protocol with the following modifications. In brief, poly-A containing mRNA molecules were purified from 5 µg total RNA using poly-T oligo attached magnetic beads. The purified mRNA was fragmented by addition of 5× fragmentation buffer (Illumina, Hayward, CA) and was heated at 94°C in a thermocycler during 5 min. The fragmentation time of 5 min is the standard time used in the protocol, which yields to fragments of ~250 bp. First strand cDNA was synthesized using random primers to eliminate the general bias towards 3' end of the transcript. Second strand cDNA synthesis was done by adding GEX second strand buffer (Illumina, Hayward, CA), dNTPs, RNaseH and DNA polymerase I followed by incubation for 2.5 h at 16°C. Second strand cDNA was further subjected to end repair, A-tailing, and adapter ligation in accordance with the manufacturer supplied protocols. Purified cDNA templates were enriched by 15 cycles of PCR for 10 s at 98°C, 30 s at 65°C, and 30 s at 72°C using PE1.0 and PE2.0 primers and with Fastart taq DNA polymerase (Roche). The samples were cleaned using QIAquick PCR purification columns and eluted in 30 μl EB (Elution Buffer) as per manufacturer's instructions (QIAGEN, CA). Purified cDNA libraries were quantified using Bioanalyzer DNA 100 Chip (Agilent Technology 2100 Bioanalyzer)