RNA Extractions and Sequencing
Samples for 3’ TagSeq were collected after two days of exposure to oral diets for three representative populations (Coffee, Dorris, Snavely), prior to the main onset of mortality. For each population, regime, and treatment, we collected nine surviving larvae and immediately stored them at -80°C until processing, and subsequently pooled larvae into three replicates of three larvae each. We extracted total RNA using a Qiagen RNeasy Mini kit according to manufacturer protocols and confirmed nucleic acid concentration and purity using a Nanodrop. We depleted remaining DNA using the Invitrogen RNAqueous Micro DNase treatment according to manufacturer protocols. We confirmed the concentration and quality of these DNA-depleted RNA samples using a Bioanlayzer (RIN > 9). We generated single indexed 3’ TagSeq libraries from 1 μg total RNA using the Lexogen QuantSeq 3’ mRNA-Seq Library Prep Kit according to manufacturer’s protocols; libraries were amplified using 15 PCR cycles. Library concentration and quality were confirmed prior to PE100 sequencing on four runs on the NextSeq platform (VANTAGE, Vanderbilt University).