RT-PCR Cloning of TRPA Genes in A. hygrophila

The RNA sample used for RT-PCR cloning of the three TRPA genes was prepared by pooling an equal amount of RNA from each of the six developmental stages. A total of 1 μg of this pooled total RNA sample was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). Then, 1 μL of the resultant cDNA sample was used as the template to RT-PCR-amplify the cDNA sequences of TRPA1, painless, and pyrexia, respectively, in a 25 μL reaction containing 12.5 μL 2 × Phanta Max Buffer, 1 μL Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China), 6.5 μL ddH₂O, and 2.0 μL of the gene-specific forward and reverse primers (10 μM) (Table S1) designed based on the contigs of each gene found in the full-length transcriptome of A. hygrophila [41 (link)]. The amplification conditions of PCR were pre-denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and elongation at 72 °C for 4 min, as well as a final elongation at 72 °C for 5 min. The obtained RT-PCR products of each gene were fractioned on a 1.2% agarose gel, eluted using a MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and cloned into pMD™19-T Vector (TaKaRa, Dalian, China). Three positive clones for each gene were sequenced (Sangon Biotech Co., Ltd., Shanghai, China).