RNA-Seq of Aedes aegypti: Midgut transcriptome
After RH treatment, mosquitoes were released into 12 x 12 x 12 cages (BioQuip) and permitted to bloodfeed to repletion (approximately 20 minutes) on a live human host (27-year-old male, leg; IRB, University of Cincinnati) or not permitted to bloodfeed but with a human leg just outside the cage. These conditions resulted in four different groups: N1, non-bloodfed/non-dehydrated (control) group; Y1, bloodfed/non-dehydrated group; N7, non-bloodfed/dehydrated group; Y7, bloodfed/dehydrated group. Three hours (1h) pbf, mosquitoes were dissected and the midguts from approximately 15 different mosquitoes were pooled and placed into Trizol (Invitrogen) held on ice. Digestion of blood occurs around 4 hours pbf (Naksathit et al., 1999) and diuresis is well underway within 2h (Drake et al., 2010; Williams et al., 1983), so dissections 3h post-bloodmeal were chosen to encompass differentially expressed genes related to altered blood digestion/water retention. Pooled midguts were homogenized (Benchmark, BeadBlaster 24), in Trizol and stored at -70C until all samples were collected. RNA was extracted with Trizol according to manufacturers protocols and cleaned with a RNeasy Mini Kit (Qiagen). DNase (Ambion, Turbo-DNA-free) was used to remove genomic DNA, cDNA libraries were generated (Illumina, TruSeq), RNA concentration was determined with a Nanodrop 2000 (Fisher), and next-generation sequencing was conducted at the Cincinnati Childrens Hospital Medical Centers DNA Sequencing and Genotyping Core.