Iso-Seq of Epinephelus awoara: whole body
RNA was converted into cDNA using the SMARTer PCR cDNA Synthesis Kit. PCR amplification was carried out with PrimeSTAR GXL DNA polymerase. After screening, the amplified products were mixed in equal molar proportions. The SMRTbell library was then constructed using the SMRTbell Template Prep Kit 1.0. This involved damage repair, end repair, and ligation of stem-loop sequencing adaptors at both ends of the cDNA fragment. Fragments that did not successfully ligate were removed using exonuclease. After purification, the library was obtained. The size of the library fragments was assessed using the Agilent 2100 Bioanalyzer. Once the library was ready, a specific concentration and volume of cDNA templates and enzyme complexes were transferred to the PacBio Sequel II instrument with Sequel II for sequencing.