Accuprime high fidelity taq system

To identify potential transcripts present in abalone gonadal tissue, methods were adapted from the PacBio Iso-seq protocolto create cDNA libraries. Ovary and testes transcriptome libraries were prepared for PacBio sequencing. RNA was extracted from H. tuberculata ovary and testes samples by cesium chloride density gradient centrifugation (MacDonald, Swift, Przybyla, & Chirgwin, 1987) (link). RNA samples were enriched for mRNA by using the Oligotext mRNA Mini Kit from Qiagen. Purified mRNA was used as the template for single stranded cDNA synthesis using the Clontech SMARTer cDNA Synthesis Kit. The cDNA was amplified by PCR using the AccuPrime High-Fidelity Taq system (Invitrogen, Carlsbad, CA, USA). Double stranded synthesis conditions were optimized with the following PCR program: °C for 2 minutes, followed by 20 cycles of 94°C for 30 s, 55 °C for 30 s, and 68 °C for 10 minutes. We used unique identifying barcoded PCR primers to amplify testes (barcode: CTGCGTGCTCTACGAC) and ovary (barcode: TCAGACGATGCGTCAT) cDNA. Because of size bias during PacBio sequencing, double stranded cDNA was fractionated using Ampure XP Beads (Beckman Coulter Life Sciences) into two fractions (Ratio of 4:1 of 0.45x : 0.6x size selection). Testis and ovary cDNA were sequenced using the PacBio RSII and was performed by the Washington State University Genomics Core.