F2T2
Total RNA was isolated from 6 testes of fish from each treatment group using TRIZOL reagent (Life Technologies, CA, USA). Three testes were pooled as one biological sample, and these two pooled replicates were used for library construction. RNA concentrations were measured using a Qubit® RNA Assay Kit on a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). A quantity of 300 ng of total RNA with an RNA Integrity Number (RIN) greater than 8, as determined by the Agilent 2100 Bioanalyzer system, was used for library construction (Agilent Technologies, CA, USA). Ten independent libraries were prepared for RNA sequencing. Briefly, the cDNA libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, USA) following the manufacturer’s recommendations. Index codes were ligated to identify the individual samples. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (Illumina, San Diego, USA) before fragmentation using divalent cations under elevated temperature in Illumina fragmentation buffer. First- and second-strand cDNAs were synthesized using random oligonucleotides and SuperScript II followed by DNA polymerase I and RNase H treatment. Overhangs were blunted by treatment with exonuclease/polymerase followed by 3?-end adenylation and ligation to Illumina PE adapter oligonucleotides. DNA fragments successfully ligated with adaptor molecules on both ends were enriched using the Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified using the AMPure XP system and quantified using the Agilent Bioanalyzer 2100 system. Before being subjected to sequencing, the libraries were normalized and pooled together in a single lane on an Illumina MiSeq platform.