2.2 Tissue collection and RNA sequencing
To facilitate the prediction of protein-coding genes, we extracted total RNA from six tissues of the leopard coral grouper, including gonad, liver, skin, spleen, muscle and fin, using TRIzol reagent (Invitrogen, CA, USA). RNA integrity and quantity was evaluated using the Agilent 2100 Bioanalyzer (Agilent, USA). The six RNA-Seq libraries, which were prepared using the NEBNext Ultra RNA Library Prep Kit (Illumina, USA) following the manufacturer’s protocol, was sequenced on a BGISEQ-500 sequencing platform (BGI, Shenzhen, China), producing 70.36 Gb raw data. The quality control of the raw reads were performed with a QC pipeline for RNA-Seq data, RNA-QC-Chain (Zhou et al., 2018), and 69.34 Gb clean RNA-Seq data were remained for further analyses (Table 1).