Illumina HiSeq 2000 paired end sequencing; D pseudoobscura reproductive tissue RNAseq after experimental sexual selection
D pseudoobscura reproductive tissue RNAseq after experimental sexual selection
All dissections were performed within 5 days in a design as balanced as logistically possible, on 5 day old flies. All flies were kept in four time-shifted incubators to avoid circadian effects on gene expression. Heads and abdomens from both sexes were dissected in 4oC RNAlater and care was taken to include antennae. The samples were stored at 4oC overnight, and then at -20oC until RNA extraction. 157 generations of artificial evolution under either enforced monogamy (M: one male housed with one female) or elevated polyandry (E: one female housed with six males) RNA was extracted using Trizol plus (Ambion) with RNA purelink columns for cleanup and DNase treatment. Ribozero (Epicentre) was used to remove rDNA. RNA extractions were assessed for quality using Nanodrop (Thermo Scientific), Bioanalyser (Agilent) and Qubit (Life Technologies). RNASeq libraries were prepared from this material using the Epicentre ScriptSeq v2 RNA-Seq Library Preparation Kit following 15 cycles of amplification, and libraries purified using AMPure XP beads (Beckman Coulter Inc.). Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyser. These final libraries were pooled in equimolar ratios. The quantity and quality of each of the pools was assessed by Bioanalyzer and subsequently by qPCR.