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.