RNA-Seq of Epinephelus awoara: whole body
For RNA sample preparation, 1 microgram (1 g) of RNA per sample was used as input material. Sequencing libraries were created using the TruSeq RNA Library Preparation Kit following the manufacturer's guidelines. Index codes were added to assign sequences to each sample. First, mRNA was purified from total RNA using magnetic beads with attached poly-T oligos. Next, first-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Subsequently, second-strand cDNA synthesis was carried out using DNA Polymerase I and RNase H. Overhanging ends of the DNA fragments were converted into blunt ends using exonuclease and polymerase activities. After adenylation of the 3' ends of DNA fragments, Illumina Adapters were ligated to prepare for hybridization. To select cDNA fragments in the preferred range of 150 to 200 base pairs, library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). PCR was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, PCR products were purified with the AMPure XP system, and library quality was assessed using the Agilent Bioanalyzer 2100 system. The index-coded samples were clustered using the cBot Cluster Generation System with the TruSeq PE Cluster Kit, following the manufacturer's instructions. The qualified libraries were sequenced on the MGISEQ2000 platform.