RNA-seq of Culicoides sonorensis: W8 Cell Line
After the supernatants were removed, the cells were lysed with Xml Trizol and RNA was collected per manufacturers instructions (Direct-zol RNA Miniprep Plus Kit; R2070). RNA was stored at -20 C. RNA quantity and quality was assessed via QubitFlex (Thermo Scientific) and NanoDrop 8000 (Thermo Scientific). Messenger RNA from extracted RNA of samples for 1, 8, 24, and 96 HPI was purified using poly-T oligo attached magnetic beads. Purified mRNA was fragmented, and first strand cDNA was synthesized via random hexamer and completed by second strand synthesis. A one step end-repair and dA-tailing was used to create 5-phosphorylated and 3-dAtailed cDNA fragments enabling direct ligation of Illumina sequencing adapters. cDNA was then size selected and amplified by polymerase chain reaction (PCR). Multiplex libraries were sequenced on the Illumina Hiseq2500 platform with at least 40-million paired-end 150BP reads per sample.