Dnase 1 recombinant enzyme

Material was collected for RNA sequencing at generations 0, 5, and 9 for the wMel-infected and tetracycline-cured control cells. Viral RNA in the cell medium supernatant was extracted using the TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s protocol. Each sample of viral RNA was solubilized in 44 µl of RNase-free water. To each sample, 1 µl of DNase I recombinant enzyme (Roche) and 5 µl of buffer was added and incubated at 37 °C for 50 min to ensure no contamination of genomic DNA. To clean up the isolated RNA for sequencing use, we used an RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and eluted purified RNA into 30 µl of RNase-free water. Samples were prepared with the Illumina whole-transcriptome library prep kit with Ribo-Zero Gold rRNA depletion, and sequencing was performed on the Illumina NextSeq 500 platform at the Australian Genome Research Facility (AGRF).

We sought to determine whether mosquitoes that varied for EIP had particular transcriptomic patterns. Mosquitoes were reared in populations and experiments carried out three generations after the original field collection. A total of four hundred 6- to 8-day-old females were fed viremic blood (Treatment = DENGUE) and assessed for EIP as per below. To control for effects of blood-feeding and age, 250 age-matched female mosquitoes were fed with nonviremic blood on the same day (Treatment = BLOOD). When a mosquito’s saliva tested positive for virus, the subsequent day the whole body of that individual was collected, homogenized, and total RNA was extracted using TRIzol reagent as per the manufacturer’s instructions (Life Technologies). RNA samples reconstituted in RNase-free water were then treated with DNase I recombinant enzyme (Roche, Basel, Switzerland) according to the manufacturer’s protocol to eliminate genomic DNA contamination. On the same day, ten whole bodies each from the BLOOD treatment were collected and extracted as above as controls to match the collection timepoint.