Magna pure 96 extraction system

DNA was extracted from frozen intact blood-fed mosquitoes using two extraction protocols, DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA) extraction protocol was used initially, and the MagNA 96 Pure DNA and Viral NA Small Volume Kit (Roche Applied Science, Penzberg, Germany) was subsequently used in an automated MagNA Pure 96 extraction system (Roche Applied Science) to increase throughput. The first batch of mosquitoes (from Homa Bay County) was extracted using DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA) extraction protocol. The 258 engorged abdomens were separated from the rest of the body using sterile dissection pins and homogenized in phosphate buffered saline (PBS). Blood was extracted following the manufacturer’s instructions. In the second batch of 214 samples from Baringo County, we used the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche Applied Science, Penzberg, Germany). Individual blood-fed mosquitoes were homogenized whole for 5 sec in 0.5 ml screw-cap tubes (Sarstedt, Newton, NC) filled with 750 mg of 2.0 mm, 150 mg of 0.1 mm zirconia/yttria stabilized zirconium oxide beads (Glen Mills, Clifton, NJ), and 350 μl of PBS before DNA extraction in an automated MagNA Pure 96 extraction system (Roche Molecular Systems, Pleasanton, CA).

Cellular RNA of diverse immune genes was quantified by RT-qPCR using the Superscript III OneStep RT-PCR (Thermo Fisher Scientific) kit and the oligonucleotides listed in Supplementary Table S1. Total RNA was isolated from compound-treated or SARS-CoV-2-infected Calu-3 cells by automated pipetting using the MagNA Pure 96 extraction system (Roche). Gene expression was calculated relative to TBP reference gene expression using the ΔΔCT method (Livak and Schmittgen, 2001 (link)). SARS-CoV-2 N gene sgmRNA was quantified in RNA extracts from infected VeroE6 and Calu-3 cells using RT-qPCR and TBP as reference gene. Gene expression was calculated relative to TBP reference gene expression using the ΔΔCT method (Livak and Schmittgen, 2001 (link)). To monitor virus growth, SARS-CoV-2 RNA was quantified in cell culture supernatants by RT-qPCR targeting the SARS-CoV-2 E gene, as described before (Corman et al., 2020 (link)).

DNA was extracted from EDTA blood using a DNA and Viral NA Small Volume kit with a MagNA Pure 96 extraction system (Roche Molecular Diagnostics^©, Pleasanton, CA) following the Pathogen Universal 200 protocol and the manufacturer’s protocol. Thirty-one of the samples had been stored at −80°C for up to 3 years, and seven had been stored at 4°C for up to 14 days. All samples were tested by pan-Plasmodium assay using in-house RT-PCR.^{19 (link)} Positive samples were further analyzed by species-specific in-house RT-PCR assays for P. falciparum,^{33 (link)}P. vivax,^{33 (link)}P. ovale,^{34 (link)} and P. malariae.^{35 (link)}Amplifications were performed using the 7500 FAST RT-PCR system (Thermofisher Scientific, Waltham, MA). The 25-μL reaction mixture contained 1× TaqMan^® Fast Universal PCR Master Mix, 2× No AmpErase^® UNG (Thermofisher Scientific), 1,000 nM of the primers, 200 nM of the probes, and 5 μL DNA eluate.
The reactions were carried out in singleplex using the following cycling conditions: 95°C for 20 seconds followed by 45 cycles of 95°C for 3 seconds and 60°C for 30 seconds. ROX (6-carboxy-X-rhodamine) was used as a reference dye.