GSM8031789: C. carpio, diploid, male, replicate_4; Cyprinus carpio; RNA-Seq
Total RNA was isolated from the gonad tissue of each fish specimen. For extraction, PureLink® RNA Mini Kit (Ambion) with Trizol reagent (Thermo Fisher Scientific) and on-column PureLink DNase treatment were used according to the manufacturer´s protocol. Reagent and buffer volumes were adjusted according to the weight of tissue entering the isolation process (30 mg on average). The final elution was performed using 100 µl of RNAse-free water in the first step and the primal eluate in the second step. The yield and concentration of RNA isolates were checked using a QubitTM 4 fluorometer (Invitrogen by Thermo Fisher Scientific) and Qubit RNA HS Assay Kit (Thermo Fisher Scientific). The quality and integrity of RNA were analysed using RNA 6000 Nano Kit on a 2100 Bioanalyser instrument (Agilent Technologies). All RNA isolates were normalized by dilution at a uniform concentration of 20 ng/µl with RNase-free water. They served as templates for DNA library preparation in twice the reaction volume recommended by the manufacturer. All samples (RNA integrity number – RIN > 7) were used for DNA library preparation. 500ng of total RNA was used for mRNA enrichment using the Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Subsequently, NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®, and NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2, New England Biolabs) were used for library preparation, with 11 PCR cycles utilized for PCR enrichment. RNA fragmentation (13 minutes at 94°C) and the size selection conditions (a bead volume of 30 µl and 15 µl for the first and second bead selections, respectively) were further modified in the protocol. The quantification of DNA libraries was performed on a QubitTM 4 fluorometer (Invitrogen by Thermo Fisher Scientific) using Qubit dsDNA HS Assay Kit, and quality and size control were performed on a 2100 Bioanalyser with DNA 1000 Kit (Agilent Technologies). Finally, amplicons were pooled in equimolar amounts. The final concentration of each library in the pool was 10 nM in the pool. Subsequently, NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® and NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2, New England Biolabs) together with spike-in RNA were used for cDNA library preparation from total RNA. The quality of prepared cDNA libraries was evaluated using a Qubit fluorometer (Thermo Fisher). The quality of cDNA libraries was visualized by a 2100 Bioanalyser (Agilent), and the libraries were were finally sequenced by Macrogen Korea on Illumina HiSeq X (one lane) in a paired-end configuration producing 150 bp long reads.