Analysis was done on three RNA replicates per time point, each consisting of RNA from 3-4 pooled brains. Total RNA was isolated from zebrafish brains using the miRNeasy Mini Kit (Qiagen Inc., Valencia, CA) following manufacturer’s instructions, and stored at -70°C. RNA quantity and quality (OD260/280 and OD260/230 ratios) were determined using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). RNA integrity was characterized using the Agilent RNA 6000 Nano Kit with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
biotin
Label protocol
RNA was processed for hybridization to Affymetrix Zebrafish Genome array through a core facility in the Functional Genomics Laboratory in the Center for Ecogenetics and Environmental Health at the University of Washington according to the manufacturer's instructions.
Hybridization protocol
The standard hybridization protocol was used as recommended by Affymetrix
Scan protocol
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000 through the University of Washington Functional Genomics Laboratory in the Center for Ecogenetics and Environmental Health.
Data processing
Bioconductor oligo package, RMA normalization. Comparisons were made using the Bioconductor limma package, fitting an ANOVA model as well as an ANCOVA model with a 3 degree of freedom spline.
