Chelex

Genomic DNA was extracted from two 3 mm punched discs of dried filter paper blood blots using the Chelex extraction protocol [25 ]. Briefly, the two punched discs for each sample were incubated in 1 mL of PBS solution for 10 minutes. The samples were washed twice with 1 mL PBS, followed by centrifugation at 14,000 rpm for 2 minutes. DNA was then extracted from the discs with 100 μL of 10% Chelex (Sigma-Aldrich, USA) in nuclease-free water by heating at 99 °C for 10 minutes, with occasional vortexing. Finally the sample was centrifuges at 14,000 rpm for 1 minute and the supernatant containing the DNA aliquoted into a new tube and stored at 20 °C for PCR.

Total genomic DNA (gDNA) was extracted from the dried blood spot (DBS) using the Chelex extraction procedure [40 , 41 (link)]. Briefly, two 3 mm discs were punched out from the dried blood spots into 1.5 mL microcentrifuge tubes containing 1 mL of 1X phosphate buffered saline (PBS). The discs were washed twice in 1 mL PBS and then boiled at 99 °C in 200 μL of 20% Chelex (Sigma-Aldrich, USA) in DNase/RNase-free water. After a final centrifugation step (14,000 x g for 1 min), the extracted DNA was transferred into a labelled 0.6 mL microcentrifuge tube and then stored at − 20 °C.

From a total of 148 isolates, the genomic DNA was extracted by boiling. Briefly, cultures were grown overnight at 37 °C in blood agar plates. For each sample, a colony was added to 100 µL of Chelex^® (6%) (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 56 °C for 15 min and then at 99 °C for 10 min. The samples were centrifuged at 13,000× g for 5 min. The supernatant was collected and stored at 4 °C until use.

DNA was extracted from the samples by using the protocol that was previously established by Walsh et al. [55 (link)]. A 5% Sigma-Aldrich Chelex^® 100 stock solution was prepared by adding 5 g of Chelex powder and 95 mL of water followed by proper vertexing. An amount of 200 microliters of the 5% Chelex was pipetted out into a 1.5 mL Eppendorf tube, and several bacterial colonies were picked by sterile wire loop and dipped into Chelex, which was followed by mixing multiple times using slow pipetting to avoid bubbling. The reagent was then incubated at a temperature of 65 °C for 8 min. The reagent was vortexed several times to ensure Chelex encountered the bacterial colonies to extract DNA. The reagent was again heated at 60 °C for 7 min and, after cooling, centrifuged at 12,000 rpm for 2 min. The supernatant was collected into separate tubes, as it contained DNA, while the pellet was discarded, as it had the remaining Chelex reagent. The collected DNA was stored at −4 °C for further use.
The S. aureus isolates were molecularly identified through PCR, with a species-specific thermonuclease (nuc) gene, as previously described by Louie et al. [56 (link)]. The primers used in the amplification of the nuc gene are given in Table 3.

DNA was extracted from each semen sample by using the protocol of Chandler et al. (2002) [21 (link)] with some modifications. Sperm from 5 straws from each treatment were thawed and washed twice with D-PBS (composition). Proteinase K (Qiagen Inc., Valencia, CA, USA), 1 M dithiothreitol (DTT) in 0.01 M sodium acetate, and 5% Chelex (Sigma Aldrich (USA) were added to the samples. The samples were gently mixed and incubated at 37 ℃. After incubation, the samples were extracted with DNA using the QIAamp^® DNA mini kit (Qiagen, Valencia, CA, USA). The procedures for DNA extraction were carried out according to the manufacturer’s instructions. Quantification of the samples was performed using a NanoDropTM 2000 (Thermo Scientific, Wilmington, DE, USA) at an absorbance ratio of 260–280 nm. The sequences of the forward and reverse primers and amplicon length are given in Table 1. GAPDH was used as an internal housekeeping gene.

The electrolytes for electrolysis
were prepared from Li₂CO₃ (99.999%, Acros Organics),
NaHCO₃ (99.7%, Sigma-Aldrich), KHCO₃ (99.95%,
Sigma-Aldrich), CsHCO₃ (99.99%, Alfa Aesar), KH₂PO₄ (99.9%, Sigma-Aldrich), K₂HPO₄ (TraceSELECT, 99.999%, Sigma-Aldrich), and Milli-Q water (≥18.2
MΩ cm, TOC < 5 ppb). The HCO₃^– electrolytes were stored with Chelex (100 sodium form, Sigma-Aldrich)
to clean the electrolyte from any metal impurities.^{17 (link)} Ni(NO₃)₂·6H₂O (99.999%,
Sigma-Aldrich), Na₂HPO₄·2H₂O
(99.5%, Merck), ethanol (puriss, Honeywell), and Milli-Q water were
used to prepare the deposition electrolyte. H₂SO₄ (95–98%, Sigma-Aldrich), H₂O₂ (35%,
Merck), and KMnO₄ (99%, Sigma-Aldrich) were used to clean
the cells. Ar (5.0 purity, Linde), CO (4.7 purity, Linde), and CO₂ (4.5 purity, Linde) were used for purging the electrolytes.

Sample preparation and MS analysis were performed as described previously (33 (link)). Briefly, P. aeruginosa was grown in MHB or MHB treated with Chelex (Sigma-Aldrich, Switzerland) under standard MIC determination conditions in microtiter plates without shaking at 37°C for 18 h. The cells from three wells were combined to yield sufficient material for proteome analysis. Three replicate samples were lysed, and the proteins were reduced with 5 mM Tris (2-carboxyethyl) phosphine hydrochloride and alkylated with iodoacetamide. The samples were diluted before digestion with trypsin at 37°C overnight. The peptides were desalted on a C₁₈ reversed-phase column and dried under vacuum. One microgram of peptide was injected into a liquid chromatography-mass spectrometer (LTQ-Orbitrap Elite). The peptides were separated using an EASY nLC-1000 system (Thermo Fisher scientific) using a C₁₈ high-performance liquid chromatography (LC) column. Tandem mass spectrometry data were exported from Progenesis LC-MS and searched against a protein decoy database of P. aeruginosa.