Fastprep 24 machine

Adult lice, one female and two males combined, were homogenized with FastPrep24 machine (MP Biomedicals, California, USA) using stainless beads. The RNA was then extracted from the homogenate using a combination of Trizol (ThermoFisher scientific, Massachusetts, USA) and RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Briefly, chloroform (0.2 ml) was added to lice homogenate prepared using 1ml Trizol for phase separation. The aqueous phase was then used for RNA isolation using RNeasy Plus Mini Kit following the manufacturer’s protocol.⁶⁸ (link) After extraction, the RNA concentrations were measured by spectrophotometry using Nanodrop ND1000 (Thermo Scientific, Massachusetts, USA). cDNA was synthesized from RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) following manufactures protocol.

For each sample, DNA was extracted from 0.25 g rhizosphere soil using the MoBio PowerSoil^TM DNA Isolation Kit (Carlsbad, CA, United States). Extractions were performed according to the manufacturer’s instructions but with the use of the MP Biomedicals FastPrep-24 machine twice for 30 s at 5.5 m.s^-1. DNA purity and concentration were determined by NanoDrop spectrophotometry (Thermo Scientific, Wilmington, DE, United States) as well as a Qubit 2.0 Fluorimeter using ds DNA HS assay kit (Thermo Fisher), respectively.

DNA isolation was performed using the ZymoBIOMICS DNA Miniprep-Kit (Zymo Research Europe, Freiburg, Germany) using 45–90 mg of well homogenized fecal material following the supplier´s instructions. For an optimal cell lysis, the extraction protocol included a 1 min bead beating step (repeated 5x, each) using a Fastprep-24 machine (MP Biomedicals, Eschwege, Germany) and bashing beads in lysis solution provided with the extraction kit. DNA purity and concentration after extraction were measured with an Implen NanoPhotometer P-Class 360 (Implen, Munich, Germany).

For each sample, total soil DNA was extracted from approximately 0.25 g homogenized soil [Qiagen DNeasy PowerSoil DNA isolation kit (Venlo, Netherlands)], according to the manufacturer’s instructions using the MP Biomedicals FastPrep-24 machine twice (30 s, 5.5 m s^–1). DNA concentrations and purity were determined as above.

Cell envelope was extracted based on a previously described method [37 (link)]. Growing mid-log cultures of S. aureus in TSB (37 °C with orbital shaking at 250 r.p.m.) were harvested and diluted as appropriate to an optical density at 600 nm of ˜0.6 and resuspended to and OD₆₀₀ of 0.6. Then, 50 ml was centrifuged at 16 100 g for 5 min at 4 °C, resuspended, and washed in 1 ml of TBS [50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.5 mM PMSF, 1 mg of iodoacetamide ml^–1). Samples were centrifuged at 16 100 g for 5 min at 4 °C, and pellets were resuspended in 1 ml of TBS. Next, 0.5 ml of suspension was added to tube containing Lysing Matrix B (MP Biomedicals) containing glass beads, which was then shaken 10 times in a FastPrep-24 machine (MP Biomedicals) set at speed 60 for 40 s. The tubes were placed on ice and allowed to cool between each cycle. Glass beads were allowed to settle, and the supernatant containing insoluble cell wall material was removed. Insoluble material was recovered by centrifugation at 16 100 g for 10 min at 4 °C and washed in 1 ml cold 50 mM Tris-HCl (pH 7.5) followed by centrifugation at 16 100 g for 10 min at 4 °C before resuspension in SDS-PAGE buffer.