Sixteen adult fish (4 to 6 months old) from each strain were randomly assigned to 1-L aquaria (22 cm long, 15 cm high, 5 cm wide), one fish per aquarium. The aquaria were delimited into six 2-cm vertical zones using thin twine tied across the front of the entire row of aquaria. Fish were then observed using a high-throughput behavioral assay to quantity fear-related behaviors. Following behavioral observations, fish were removed from the individual tanks and temporily placed in one of sixteen 3-L group tanks. On the sampling day, each fish was anesthetized in 170 mg L−1 tricaine methanesulfonate (MS222, Western Chemical Inc., Ferndale, WA), briefly blotted on a paper towel, and rapidly measured for standard length and body mass. The brain was then quickly removed and homogenized in TRIzol (Invitrogen, Carlsbad, CA). Brains from all individuals in a tank were homogenized together, for a total of two biological replicate pools per sex per strain (16 microarrays total).
Growth protocol
Prior to the experiment, all zebrafish were reared in a recirculating zebrafish facility at the University of Idaho, designed by Aquaneering Inc. (San Diego, CA). Fish were feed twice daily with a combination of commercial flake food and Artemia nauplii. Temperature of the facility was maintained at 28ºC with a constant 14 h light: 10 h dark cycle. These rearing conditions were maintained during the behavior assays.
Extracted molecule
total RNA
Extraction protocol
Total RNA was then extracted using the TRIzol method following manufacturer’s protocol (Invitrogen).
Label
biotin
Label protocol
For each array, 10 ug of total RNA were converted to cDNA. Biotinylated cRNA was then produced in vitro using the GeneChip expression 3′ amplification one-cycle target labeling kit (Affymetrix, Santa Clara, CA, USA).
Hybridization protocol
Affymetrix Zebrafish Genome Arrays (~14,900 transcripts) were hybridized with fragmented biotinylated cRNA for 16 h at 45°C with constant rotation (45 rpm), and processed using the Affymetrix GeneChip Fluidic Station 450. Streptavidin-conjugated phycoerythrin (SAPE) was used for staining, followed by amplification using a biotinylated anti-streptavidin antibody. This was followed by another round of SAPE. All microarray procedures were performed at the Genomics Core Facility of the Center for Reproductive Biology at Washington State University (Pullman, WA).
Scan protocol
All microarrays were scanned using a GeneChip Scanner 3000 (Affymetrix).
Description
Gene expression data from brain of domesticated TM1 zebrafish strain
Data processing
CEL files containing raw data were then processed and analyzed using R software and Bioconductor packages. Microarray hybridization data were examined for physical anomalies on the chip by pseudochip and residual error visualizations. Quality assurance of microarray data was completed using the affyQAReport function from the Bioconductor package affyQCReport. The arrays were then pre-processed using the Robust Multi-array Average (RMA) procedure using the affy package. Next, unexpressed and low variability genes were removed by unbiased filtering. Affymetrix present-marginal-absent (PMA) calls were determined using a P-value cut off for absent of greater than 0.04 and present less than 0.04; marginal calls were treated as absent. Unexpressed genes were then defined as having a signal less than the expression value at which 99% of genes were called as absent across all samples. A filter on interquartile range was also applied to remove genes with low variability. Genes with an interquartile range of less than 0.5 across all chips in the experiment were excluded, reducing the dataset further to 7,958 genes. Signal intensities were also examined at the probe level to identify single feature polymorphisms (SFPs), differences at the probe level due to genetic polymorphisms rather than expression differences, which may also impact computed expression values. Briefly, in R using previously described methods, the RMA normalized expression estimate for each probe set was subtracted from background corrected and normalized expression levels at individual probes within the probe set. Normalized residuals were analyzed using significance analysis of microarrays (SAM) within the siggenes package to detect features with a significant effect for strain (FDR adjusted < 0.01). A total of 3,199 genes with significant SFPs were then removed from the analysis.
