Sample processing for RNAseq and quantitative analysis of transcriptomes
mRNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA), and purified to remove DNA contamination using the TURBO DNA-freeTM kit (Invitrogen, Carlsbad, CA). mRNA sample were sent to the Israel National Center for Personalized Medicine (INCPM) at the Weizmann Institute of Science (Rehovot, Israel), where quality was determined on TapeStation Agilent 2200 system before library preparation and sequencing on an Illumina Hi-Seq 2500 device. Raw sequencing single ended data files were retrieved for trimming and adapter removal using Trimmomatic (v. 0.39, Bolger et al. 2014). After passing quality control assessment with FASTQC software (v. 0.11.9, Andrews et al. 2010), the fastq files were mapped against the O. niloticus reference genome (GCF_001858045.2) using STAR software (v. 2.7.3a, Dobin et al. 2013). The resulting BAM files from the mapping were processed with the package HTSeq (v. 0.11.1, Anders et al. 2014) in R (v. 3.6.3, R Core Team 2020) for obtaining the gene counts which were then submitted to the DEseq package (v. 1.39.0, Anders et al. 2010) in R (v. 3.6.3, R Core Team 2020) for retrieving up- and down-regulated genes, according to the experimental design. Significance was considered after adjusted p-value less than 0.05. Significant differentially expressed genes (DEG) were evaluated for their gene ontology (GO) annotations. All transcriptomics data are available at NCBI (https://submit.ncbi.nlm.nih.gov/subs/biosample/SUB8325839/).