Feature extraction software v9.5

Global cDNA Whole-Human Genome 4 × 44 K Microarray (Agilent) gene expression profiling was performed on the same tissue specimens previously mentioned, as described elsewhere^{36 (link)}. Briefly, 100 ng of mRNA were reverse transcribed using Superscript II reverse transcriptase (Invitrogen Canada, Burlington, ON) while incorporating Cy3-dCTP or Cy5-dCTP (NEN, Boston, MA, USA). Raw microarray image files were extracted using Agilent Feature Extraction Software (v9.5). Microarray data were pre-processed and normalised using agilp v3.4.0 (bioconductor v3.3); only probes that matched to a gene symbol were included. In case of multiple ID mappings, the average was calculated between the matching probes, with no filtering on variance or expression threshold. Six samples (4_D1, 4_O1, 8b_T, 10_D1, 10_D2 and 22_D1) were excluded, after QC, and were defined as outliers using a sum of standard error boxplot.

Gene expression analysis was performed on fish at five stages: recrudescence (n = 5), developing (n = 6), pre-spawning (n = 7), spawning (n = 7), and post-spawning (n = 6) (n = 31 microarrays). Microarray hybridizations were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol using Cyanine 3 (Cy3) and 100 ng total RNA per sample was used for the production of cDNA and labeled/amplified cRNA as per the Agilent Low RNA Input Fluorescent Amplification Kit. Labeling methods followed those previously described [18 (link)]. Each RBD testis sample showed a specific activity >6.0 pmol Cy3/mL and amounts were adjusted to a final mass of 0.825 μg for 8x15K microarray hybridizations. Fragmentation of the cRNA, hybridization, and slide washes after the 17 h hybridization followed instructions outlined by Agilent (Gene Expression Hybridization Kit). An ozone barrier slide was used to cover microarrays before scanning. Microarrays were scanned at 5 μm with the high density Agilent DNA Microarray Scanner (Agilent Technologies). Agilent Feature Extraction Software (v9.5) scanned arrays with a full dynamic range. The quality of microarray data was evaluated by manual inspection and the quality control report generated by Feature Extraction Software (Agilent Technologies). Each microarray was deemed to be of high quality based on visual inspection.