RNA extraction and sequencing

We generated three biological replicates per species and tissue type from pooled individuals (1–9 individuals per replicate, a total of 516 individuals, in 150 replicates in total (including the virgin sexual females); see Supplementary Data ⁸). To extract RNA, samples were flash-frozen in liquid nitrogen followed by addition of Trizol (Life Technologies) before being homogenised using mechanical beads (Sigmund Lindner). Chloroform and ethanol were then added to the samples and the aqueous layer transferred to RNeasy MinElute Columns (Qiagen). RNA extraction was then completed using an RNeasy Mini Kit following the manufacturer’s instructions. RNA quantity and quality was measured using NanoDrop (Thermo Scientific) and Bioanalyzer (Agilent). Strand-specific library preparation and single-end sequencing (100 bp, HiSeq2000) were performed at the Lausanne Genomic Technologies Facility.

The 150 libraries produced a total of just under 5 billion single-end reads. Six whole-body and six tissue-specific libraries produced significantly more reads than the average for the other samples. To reduce any influence of this on downstream analyses, these libraries were sampled down to approximately the average number of reads for whole-body or tissue-specific libraries respectively using seqtk (https://github.com/lh3/seqtk Version: 1.2-r94).