Mini beadbeater 8 instrument

Bacterial pellets were re-suspended in RLT buffer (QIAGEN, Hilden, Germany), transferred to screw-cap tubes containing 250 mg of 0.1 mm zirconia/silica beads (BioSpec Products, Bartlesville, OK, USA) and disrupted by bead beating for 1 min for four times, each followed by 1 min on ice, using a Mini-Beadbeater-8 instrument (BioSpec Products, Bartlesville, OK, USA). RNA was then extracted with RNeasy Protect Bacteria Mini Kit (QIAGEN, Hilden, Germany), following manufacturer’s instructions. Integrity of the RNA sample was verified on 1.5% denaturing agarose gel. Total RNA was treated with Turbo DNA-free kit (Thermo Fisher Scientific, Waltham, MA, USA), RNA concentration and purity were measured using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and 1 µg of RNA was reverse transcribed with SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) using random hexamers, according to the manufacturer’s protocol.

Plaque assays were performed as previously described [76 (link)]. NIH 3T12 cells were plated in six-well plates 1 day prior to infection at 2×10⁵ cells per well. Organs were subjected to 4 rounds of mechanical disruption of 1 min each by using 1.0mm zirconia-silica beads (Biospec Products, Bartsville, OK) in a Mini-Beadbeater-8 instrument (Biospec Products). Serial 10-fold dilutions of organ homogenate were plated onto NIH 3T12 monolayers in a 200-μl volume. Infections were performed for 1h at 37°C with rocking every 15 min. Immediately after infection plates were overlaid with 1.5% methylcellulose in complete DMEM. After 10 days, cells were stained with 0.12% (final concentration) Neutral Red. The next day, methylcellulose was aspirated, and plaques were enumerated. The sensitivity of the assay is 50 PFU/organ.