title: GSM8625522: Midgut_KUMstrain, MockBlood_Day7, scRNAseq; Aedes aegypti; RNA-Seq
library_construction_protocol: A cold dissociation method was used for the mosquito midgut and fat body, adapting protocols originally developed for dissociating Anopheles sp. midgut into single-cell suspensions suitable for scRNAseq (DOI: dx.doi.org/10.17504/protocols.io.j8nlke246l5r/v1, remove the private link prior to publication). Mosquitoes were dissected on ice and pools of 7 organs collected in a drop of ice-cold SF900 III media (Sigma-Aldrich). Each organ was sectioned into 3 smaller pieces using spring scissors (Fine science tools). Sectioned organs were transferred immediately to a chambered coverglass well (Thermo scientific) containing 500ul of dissociation media (SF900 III media supplemented with 10mg/ml Bacillus licheniformis protease (Sigma-Aldrich) and 25 unit/ml DNaseI (Sigma)) for 15 minutes in the dark on ice. Tissues were then gently triturated using 15 back-and-forth pipetting motions. After 5 minutes, 100uL of supernatant containing dissociated cells was collected and transferred to a collection tube containing 45 mL of SF900 III media on ice. The dissociation well was supplemented with 100uL of fresh dissociation media and incubated for 10 min in the dark on ice. Tissues were triturated again, and 100uL of supernatant transferred to the collection tube. Three to four more dissociation rounds were performed until only single cells remained.  The collection tube was then spun at 300g for 15 min at 4°C. The pellet was resuspended gently in 900 ul ice-cold SF900 II media and filtered through Flowmi cell strainers (Sigma) with a 40 um porosity for midguts and 70 um porosity for fat bodies. The resulting cell suspension was spun at 300 g for 5 min at 4°C and resuspended in 30 ul of PBS 1x-BSA 0,04%. Library was performed according to manufacter's instruction (Single Cell 5' Kit v2 dual index,10x Genomics).