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).