Precellys homogenization tube

Each replicate was transferred to a 2 ml Precellys^® homogenization tube (Bertin Instruments, France), containing a mix of 1.4 mm and 2.8 mm ceramic beads and homogenized in 600 µl of TRIzol^® reagent (ThermoFisher Scientific, USA) with a Precellys^® 24 Tissue Homogenizer (Bertin Instruments, France), using 2 × 15 s of homogenization at 1400 x g with a 10 s. break between. For RNA extraction, a Phenol/Chloroform‐based single‐step extraction in combination with a spin column based on solid phase extraction (Direct‐zol™ RNA MiniPrep Kit, Zymo Research, USA) was used. Genomic DNA was removed by DNase I digestion on column as part of the RNA extraction kit and total RNA was eluted in ultra‐pure water. Samples were sent to GeT‐PlaGe core facility in dried‐ice for RNA sequencing.

Lung toxin/rnaIII transcript levels in mice were examined 16 h after MRSA inoculation, with or without Pi Sup. Each group of animals was sacrificed at specific time intervals; the lungs were removed and placed into a 7-ml Precellys homogenization tube (CK28) with 2 ml of RNA later, and then processed twice at 5500 rpm for 20 s. The homogenized suspension was transferred into 2-ml Precellys homogenization tubes (CK01; ceramic beads of 0.1 mm; Bertin Technologies), and again processed twice at 6000 rpm for 30 s. Using 100 μl of the homogenized suspension, total RNA was extracted using NucleoSpin RNA kits according to the manufacturer’s instructions. After RNA extraction, cDNA synthesis was carried out, and mRNA transcript levels of hla, spa, tst, pvl, rnaIII, and gyrB were determined by quantitative real-time PCR as described above.