Beadbeater machine

The detection of viral RNA in each individual was done by qRT-PCR [13 (link)]. Mosquitoes were kept under laboratory conditions. At three time points—3, 7 and 14 days after intrathoracic infection (d.p.i.)—they were individually stored in tubes and cryopreserved in a −80°C freezer for the preservation of viral RNA. After the collection and storing of all mosquitoes in all doses and time points, Ae. aegypti were individually assayed for viral load.
For RNA extraction each mosquito was, separately, put into a 2.0mL vial-tube with one glass bead 2mm and 50 μL of PBS Buffer one-fold. The mosquitoes were then beaten for 90 seconds on Bead-beater machine (Biospec Products). After that, we performed the RNA extraction using High Pure Nucleic Acid kit (Roche) commercial kit following the manufacturer instructions. The RNAs were then quantified on Nanodrop Spectrophotometer (Thermo Scientific) and diluted to 50ng/μL. For virus detection, we used a pair of primers DENV-Forw: 5’-AAG GAC TAG AGG TTA GAG GAG ACC C- 3’ and DENV-Rev: 5’- CGT TCT GTG CCT GGA ATG ATG- 3’ and DENV: 5’-HEX/AAC AGC ATA TTG ACG CTG GGA GAG ACC AGA/3BHQ_1/3’ probe that amplifies a 109pb fragment.

RNA isolation, sample preparation, and sequencing was conducted at the University Medical Center Groningen in Groningen, the Netherlands. Each sample was homogenized with zirconia/silica beads in the BeadBeater machine (BioSpec products, Inc.). After homogenization, total RNA was extracted and purified using an RNeasy microkit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. An initial quality check of the samples by capillary electrophoresis and RNA quantification for each sample was performed using the LabChip GX (PerkinElmer, Waltham, Massachusetts, USA). Samples with a minimum amount of 7 ng non-degraded RNA were selected for subsequent sequencing analysis. Sequence libraries were generated using the TruSeq RNA sample preparation kit from Illumina (San Diego, USA) using the Sciclone NGS Liquid Handler (Perkin Elmer). To remove contamination of adapter-duplexes, an extra purification of the libraries was performed with the automated agarose gel separation system Labchip XT (Perkin Elmer). The obtained cDNA fragment libraries were sequenced on an Illumina HiSeq2000 using default parameters (single read 1x100bp) in pools of 10 or 11 samples. Processing of the raw data, including a demultiplexing step, was performed using Casava software (Illumina) with standard settings.

Fecal pellets were harvested from 2-month-old female TLR5^−/−NOD and TLR5^+/+NOD mice and homogenized in sterile PBS using a bead beater machine (BioSpec). Fecal material was then centrifuged for 2 mins at low speed (52 × g) to remove dietary residue. The supernatant was transferred to a new tube and spun. After washing the pellet two more times, the combined supernatant was centrifuged at 469 × g to remove mammalian cells. Bacteria in the supernatant were pelleted by centrifugation at high speed (1876 × g, 5 min) and resuspended in sterile PBS. Bacterial concentration was measured with a spectrophotometer (Bio-Rad) and heat-inactivated at 90°C for 20 min. The heat-inactivated bacteria (108) were co-cultured with splenocytes (2 × 10⁶/ml) from TLR5^−/−NOD or TLR5^+/+NOD mice for 12 hours prior to intracellular cytokine (ICC) staining.