Tissue collection and processing
We collected brain tissue from the Aripo and Quare lineage males described above in 2013 and 2015, respectively. To standardize any effects of recent experience, behavior, and circadian rhythm on gene expression, we collected whole brains within 10 minutes of lights-on. We interpret our transcriptional data as baseline, in the sense that fish were minimally stimulated prior to tissue collection, and expression levels should therefore reflect genetic background and developmental experience more strongly than responses to immediate environmental context. Fish were anesthetized by immersion in ice water followed by rapid decapitation. Whole brains were removed, flash frozen in liquid nitrogen, and stored at -80°C until further processing. Tissue collection took <2 minutes, rapid enough to minimize changes in gene expression responses to handling and dissection.
We extracted total RNA from brain tissue using the Qiagen RNeasy Lipid Tissue Mini Kit (Qiagen, Germany) following manufacturer guidelines. We prepared separate sequencing libraries for each individual using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Massachusetts, USA) following manufacturer instructions. Libraries were sequenced on an Illumina HiSeq 2000 at the Florida State University College of Medicine Translational Science Laboratory (Tallahassee, Florida) in May 2014 (Aripo dataset) and January 2016 (Quare dataset). For the Aripo dataset, 40 samples (N=10 per group) were combined with unique barcodes into eight samples per pool and each pool was sequenced on a single lane. For the Quare dataset, 60 samples (N=12-14 per group) were combined into three pools with 20 samples per pool and each pool was sequenced in two separate lanes. Blocks of individuals run in the same week were processed and sequenced together, and experimental groups were balanced across sequencing lanes.