Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: For RNA-seq library construction, 2 μg total RNA per sample was treated with DNase I (New England BioLabs) to remove possible contamination with genomic DNA. Then poly (A+) mRNA was purified from the total RNA using the Dynabeads mRNA Purification Kit (Invitrogen), followed by fragmentation using the RNA fragmentation kit (Ambion). Next, the first cDNA strand was synthesized using random hexamer primers and reverse transcriptase (Invitrogen), and second-strand cDNA was synthesized using DNA polymerase I (New England BioLabs). After that, the synthetic double-stranded cDNA was end repaired, followed by 3’-end addition of dA, adapter ligation and size selection according to Illumina’s protocols. Finally, the selected products were amplified by 15 PCR cycles to yield the RNA-seq libraries. All RNA-seq libraries were subjected to 90 bp paired-end sequencing on an Illumina HiSeq 2000 platform. We analyzed brains from all eight individuals separately, but female ovaries were only large enough for individual analysis in breeders, whereas subordinate ovaries were so small that 3–5 of them had to be pooled for gene expression analyses. The testes of breeding and non-breeding males were not visually different, but breeder status was known because these males had previously sired pups. RNA and DNA were isolated as described in Kim, et al. Nature (2011).