title: GSM8282824: Control; Aedes aegypti; RNA-Seq
library_name: GSM8282824
library_construction_protocol: Female mosquito midguts were dissected after 4 days of either blood feeding or infection. Each midgut was finely chopped into small pieces using a needle and immediately transferred into an Eppendorf tube containing 200 μl of cold Schneider's Drosophila Medium with 0.2 mM ethylenediaminetetraacetic acid (EDTA) to minimize exposure to room temperature. A total of fifty midguts from each group were collected and incubated with 200 μl of a 2 mg/ml elastase/PBS solution on a shaker at 37°C for 30 minutes. To halt the digestion reaction and prevent cell aggregation, 400 μl of 2% bovine serum albumin (BSA) in PBS was added. The cell suspension was then passed through a 40 μm cell strainer and loaded onto the top of an Optiprep (Axis-Shield) density gradient with a density of 1.216 g/ml. Viable cells were isolated from the top layer of the sample after centrifugation at 800g for 20 minutes. Cell viability and count were determined through 0.4% trypan blue staining and cell counting using a hemocytometer. Reverse transcription and library preparation were conducted at KMSR using the Chromium Next GEM Single Cell 5' GEM Kit v2. Both poly-dT primer and the Zika virus NS5 RT primer were employed in the generation of the cDNA library, facilitating subsequent processing with the 10x Genomics.