Proteinase k solution

Overnight culture of S. caprae strains grown in 10 ml of TSB at 37 °C was harvested by centrifugation with 8,000 rpm for 5 min. The pelleted cells were then resuspended in 3 ml of TE buffer containing 150 μg of lysostaphin (Sigma-Aldrich. Co., LLC., St Louis, MO) and 100 μg of RNase A (Sigma-Aldrich. Co., LLC.). Following a 2-h incubation at 37 °C, 525 μl of 10% SDS and 37.5 μl of Proteinase K solution (Qiagen, Hilden, Germany) were added to the cell lysate. The mixture was then incubated at 56 °C for 2 h. After incubation, one volume of tris-saturated phenol (pH 8.0) was added, and the resulting solution was mixed well by inverting the tube for 5 min. After centrifugation, the aqueous phase was transferred into a new tube. An equal volume of chloroform: isoamylalcohol in the ratio of 24: 1 (vol/vol) was added and mixed well. Following centrifugation, the aqueous phase was transferred into a new tube and a 1/25 volume of 5 M NaCl and 2.5 volume of ethanol were added. After centrifugation, the DNA pellet was washed by 80% ethanol and dried briefly. TE buffer was added to dissolve the DNA. The extracted DNA was treated with RNase Again and purified with a DNeasy Blood and Tissue Kit (Qiagen).

To isolate total RNA, an approximately equal number of the cells (4 [OD₆₀₀ × mL]) (e.g., 4 mL culture was collected when OD₆₀₀ = 1) were collected from the bioreactor at the exponential and the stationary phase (see Figure 1 for details of the sampling points). To preserve RNA integrity and transcriptome profile, the collected samples were immediately mixed with 2 volume of RNAProtect™ Bacteria reagent (Qiagen) and then incubated at room temperature for 20 min. After being harvested at 5000 × g for 10 min, cells were resuspended in 100 μl of lysozyme solution (15 mg mL⁻¹ in 30 mM Tris∙Cl, 1 mM EDTA, pH 8.0), with the addition of 10 μl of proteinase K solution (Qiagen). The cells were incubated for 20 min at room temperature with periodic tapping every 3 min. 1 mL of RiboEX solution (GeneAll, Seoul, South Korea) was mixed with the enzyme-treated cells to extract total RNA according to the manufacturer’s protocol.

SARS-CoV-2 RNA was searched in all the cases on sections from olfactory bulb and tract, postrolandic cortex and rostral medulla as previously described (10). FFPE 10 micron sections were pre-treated using 160 µL Deparaffinization Solution (Qiagen, Hilden, Germany) with 180 μL of tissue lysis buffer (ATL buffer, Qiagen) and 20 μL of protease (Proteinase K solution, Qiagen, Germany). Tissues were incubated overnight at 56 °C and one-hour at 90 °C. Then extraction and amplification of nucleic acids were performed using ELITeInGenius^® instrument with ELITeInGenius SP 200 and SARS-CoV-2 ELITe MGB Kit (ELITechGroup, Milano, Italy). The tissue viral load was reported as number of copies/microgram RNA. Quantification of RNA was performed using Qubit Assay (Invitrogen by ThermoFisher). Positive results below the lower limit of quantification (2 copies/reaction) were reported as <15 copies/microgram RNA. The identification of key mutations in SARS-CoV-2 Spike Protein gene (E484K, N501Y, HV69-70 del, L452R, K417T, K417N, W152C) was performed with Allplex SARS-CoV-2 Variants I and II Assay, Seegene.

DE (calcined powder), acetic acid (99.7%), dimethyl suberimidate dihydrochloride (DMS, 98%), lysozyme solution (30 mg/mL in distilled water), (3-aminopropyl) triethoxysilane (APTES, 99%), 3-aminopropyl(diethoxy)methylsilane (APDMS, 97%), [3-(2-aminoethylamino) propyl] trimethoxysilane (AEAPTMS, 80%), and N1-(3-trimethoxysilylpropyl) diethylenetriamine (TPDA, technical grade) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Ethanol (99%) and phosphate-buffered saline (PBS, 10×, pH 7.4) were ordered from Thermo Fisher Scientific, Waltham, MA, USA. DNase solution (1500 Kunitz units RNase-free DNase I) and Proteinase K solution (>600 mAU/mL) were purchased from Qiagen, Germany. Brucella agar was purchased from MB cell, Seoul, Korea. 5% defibrinated sheep blood was purchased from Kisan Bio, Seoul, Korea. nutrient broth and trypticase soy broth were purchased from BD Difco, Sparks, MD, USA. Sabouraud dextrose agar with chloramphenicol was purchased from Hardy Diagnostics, Santa Maria, CA, USA. All the regents were used without any further purification.

Total DNA and RNA were extracted using the DNeasy Blood and Tissue Kit (Qiagen) and RNeasy Mini Kit (Qiagen), respectively, according to the manufacturer’s instructions. The DNA samples were treated with RNase A solution (Qiagen) and Proteinase K Solution (Qiagen) at 65 °C for 10 min and RNA samples were treated with DNase I (Qiagen) for 20 min at room temperature.

Hyflo Super Cel (Diatomaceous earth), 3-aminopropyl(diethoxy)methylsilane (APDMS, 97%), dimethyl suberimidate dihydrochloride (DMS, 98%), lysozyme solution (50 mg/mL in distilled water), sodium hydroxide solution (50% in H₂O), N-Acetyl-L-cysteine (NALC, 99%), sodium citrate, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris-HCI (pH 8.0), distilled water (DNase/RNase-Free), and EDTA (pH 8.0) were purchased from Invitrogen (Waltham, MA, USA). Proteinase K solution (>600 mAU/mL) was purchased from Qiagen (Hilden, Germany). Absolute ethanol was purchased from Merck (Whitehouse Station, NJ, USA). Phosphate-buffered saline (PBS; 10×, pH 7.4) was purchased from Gibco (Grand Island, NY, USA).

Upon receipt of the tissue from the NeuroBioBank, DNA degradation was evaluated by isolating DNA from the tissue using the Qiagen EZ1 Advanced XL machine (Qiagen Catalog No. 9001875) and EZ1 DNA Tissue Kit (Qiagen Catalog No. 953034). Briefly, tissue was dissected with a scalpel on a cold block to produce approximately 34 mg of tissue. Then, 190μl of G2 buffer (Qiagen Catalog No. 1014636) and 10μl Proteinase K solution (Qiagen Material No. 101406) was added to the tissue. These samples were then vortexed for 15 seconds, briefly centrifuged for 15 seconds at maximum speed on the benchtop centrifuge and placed onto the thermo-mixer at 56°C until the tissue was dissolved (approximately 2 hours). Subsequently, all samples were pipette mixed, briefly centrifuged for 15 seconds at maximum speed on the benchtop centrifuge, and loaded onto the Qiagen EZ1 machine for extraction, with use of the EZ1 DNA Reagent Cartridge for tissue (from Qiagen Catalog No. 953034). The elution value was set as 200 μl for each of the extraction methods, and once extracted, all samples were stored at −20°C until quantification was carried out. Genomic DNA Screen Tape (Agilent Catalog No. 5067–5365) was used to determine DNA length. Tissues with fragmented DNA were not selected for further studies.