Transcriptome of female and male Xiphophorus maculatus Jp 163 A
cDNA synthesis was performed using SMART cDNA amplification technique with some modifications. The first-strand cDNA was generated by utilizing the 5’Smart Oligo, 5'-AAGCAGTGGTAACAACGCATCCGACGCGGG-3' and 3'Oligo dT SmartIIA, 5'-AAGCAGTGGTAACAACGCATCCGACTTTTTTTTTTTTTTTTTTTTTT-3'. The reaction was carried out in a 20 µl system containing 2 µg of total RNA. 20 µl of the first-strand cDNA synthesis product was used as template for the 1st run of long-distance-PCR (LD-PCR) with optimized cycle number to amplify cDNA for normalization. SmartIIA 5'-AAGCAGTGGTAACAACGCATCCGAC-3' was used as primer in LD-PCR. The volume of each reaction was 100 µl. The products of the 1st run of LD-PCR were purified using QIAquick PCR Purification Kit. Equal amount of the purified LD-PCR products from each RNA sample was mixed for normalization. TRIMMER cDNA Normalization Kit (Innovative Biotechnology Company, Moscow, Russia) that uses duplex-specific nuclease (DSN) treatment was used for normalization reaction. The normalized cDNA was used as template for the 2nd run of LD-PCR. The SMART II was used as the primer for LD-PCR. The products were purified using QIAquick PCR Purification Kit for sequencing.