RNA-seq of Culex pipiens mosquitoes infected with West Nile virus: legs 14 days post exposure: Sample Culex pipiens legs
RNA was extracted from 50 µl of sample (i.e. serum, homogenate, or supernatant) using the using the Mag-Bind Viral DNA/RNA 96 kit (Omega Bio-Tek) on the KingFisher Flex Magnetic Particle Processor (Thermo Fisher Scientific) and RNA eluted in 50 µl of nuclease-free water, 1 µl of the RNase inhibitor SUPERase-In (Ambion) and 0.5 µl of linear polyacrylamide (Ambion), followed by Turbo DNase (Ambion) treatment. Total RNA was amplified using the NuGEN Ovation RNA-Seq System V2 and cDNA amplicons were sheared using the Covaris S2 Focused-ultrasonicator. Sequencing libraries were prepared from 22-100 ng of sheared cDNA using NuGEN’s Ovation Ultralow Library Kit according to the manufacturer’s recommendations. Agencourt RNAclean XP beads were used for all purification steps. 