title: RNA-Seq of HUVEC cells: control 2
design: Primary Umbilical Vein Endothelial Cells were grown according to the manufacturers instructions. Cells were allowed to grow to 50% confluence overnight at 37C, 5% CO2. An. darlingi or An. albimanus SGE in a final concentration of 5ug/mL was prepared in endothelial media (Lonza) and filtered to avoid bacteria contamination. After a 24h incubation period, cells were harvested, and total RNA was extracted using QuickRNA (Zymo). Library construction was prepared following Illuminas TruSeq-stranded-mRNA sample preparation protocol. RNA integrity was checked with Agilent Technologies 2100 Bioanalyzer. Poly(A) tail-containing mRNAs were purified using oligo-(dT) magnetic beads with two rounds of purification. After purification, poly(A) RNA was fragmented using divalent cation buffer in elevated temperature. Quality control analysis and quantification of the sequencing library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-ended sequencing was performed on Illuminas NovaSeq 6000 sequencing system.