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/).