Agilent bioanalyzer rna pico chip

Collected mosquitoes were pooled per species, season, and collection location (indoor/outdoor) to a total of 10 mosquitoes maximum per minipool, and homogenized with 500 µL of PBS using a MagnaLyser version 1.1 (Roche, Mannheim, Germany) at 6000 rpm for 1 min. Crushing material was centrifuged for 2 min at 12,000× g and 4 °C, then 167 µL of supernatant was transferred individually to 835 µL of RNA later solution (Invitrogen). The mixture was incubated overnight at 4 °C and stored at −80 °C until shipment to Institut Pasteur in Paris. According to the mosquito species season and collection location (indoor/outdoor), minipools were combined to form large pools that contained a maximum of 100 mosquitoes per pool (Table S1). A total of 6646 mosquitoes were selected and subsequently distributed across 103 large pools. Overall, total RNA was extracted from the 103 large pools of mosquitoes in a Biosafety Level 3 (BSL-3) laboratory using the Maxwell RSC simply RNA tissue kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. RNA extracts were quantified with the Qubit RNA High sensitivity assay (Invitrogen, Waltham, MA, USA) and analyzed using an Agilent BioAnalyzer RNA pico chip (Agilent, Waldbronn, Germany).