Omni bead ruptor 12

Using a TissueLyser II, freeze-dried, powdered muscle samples were disrupted in ice-cold sucrose lysis buffer by 5 mm stainless steel beads during 3 × 2 min cycles at 20 Hz. A 10-fold volume excess of ice-cold sucrose lysis buffer was used to lyse each sample. Resulting mixed muscle lysates were centrifuged for 10 min at 8,000 g (4°C) and the supernatant was stored at −70°C before further analysis. Snap frozen liver tissue (∼30 mg tissue) was lysed in 400 uL RIPA buffer plus PhosSTOP and cOmplete ULTRA (1 tablet in 10 mL RIPA buffer). Lysates were disrupted in Fisherbrand Pre-Filled Hard Tissue Ceramic bead mill tubes (2 mL capacity, 2.8 mm particle size) using Omni Bead Ruptor 12 for 3 cycles of 4 m/s speed, 20 s. Lysate suspension was centrifuged at 10,000 g for 5 min to pellet debris and lysate supernatant was stored at −70°C before further analysis.

Macrocolony biofilms were grown for 3 days on 1% tryptone and 1% agar. Each biofilm was transferred to a 2-ml tube with a sealing O-ring (Benchmark Scientific D1031-T20) containing 1 ml of phosphate-buffered saline and 0.5 g of ceramic beads (Fisher Scientific 15-340-159). Samples were homogenized using an Omni Bead Ruptor 12 (Omni 19–050) on the “high” setting for 90 s at 4°C. Samples were left on ice for 10 min to allow bubbles to clear, and then diluted in PBS by a factor of 10⁻⁶. Ten microliters were spotted on 1% tryptone, 1.5% agar plates, which were tilted vertically to allow the spot to run down the plate and thereby increase the growth area. After 16 h of growth at 37°C, single colonies were counted and recorded.

RNA was prepared according to the manufacturer's instructions using the Qiagen RNeasy Micro RNA kit (Cat# 74004; Qiagen, Hilden, Germany) for brain slices and isolated microglia, or using the Qiagen RNeasy Mini RNA kit (Cat# 74104; Qiagen, Hilden, Germany) for brain tissue. For the homogenization step, brain slice samples were homogenized using the Omni Bead Ruptor 12 (Cat# 19-050A; Omni International, Kennesaw, GA) in 400 μL RLT lysis buffer with 350 μL used as input for RNA isolation; and for whole cerebrum samples homogenization was performed in 5 mL QIAzol lysis reagent (Cat# 79306; Qiagen, Hilden, Germany) with 200 μL used as input for RNA isolation. The purified RNA was eluted in 14 μL RNAase-free water. QC was performed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) using the RNA 6000 Pico Kit (Cat# 5067-1513; Agilent Technologies, Santa Clara, CA). RIN scores for more than 90% samples tested were eight or higher (>75% were nine or higher). Quantitation was performed using the NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA) or Quant-iT RiboGreen RNA Assay Kit (Cat# R11490; Thermo Fisher Scientific, Waltham, MA), for tissue and microglia samples, respectively.