Fastprep 24

Fungal biofilm recovered from a single well of a six-well plate was centrifuged and resuspended in 800 μL of Fenzol (A&A Biotechnology, Gdańsk, Poland). The cells were placed in a tube containing 0.8 mL of zirconia beads (0.1 mm in diameter) and disrupted in a bead-beater (FastPrep24 instrument, MP Biomedicals, Santa Ana, CA, USA MP Biomaterials) at the highest power by two 30 s pulses, separated by 5 min of chilling on ice. All other steps were performed with the Total RNA Mini Kit (A&A Biotechnology, Gdańsk, Poland) according to the manufacturer’s instructions. Genomic DNA was eliminated with deoxyribonuclease I (Sigma Aldrich, Dorset, UK). The DNA removal was accomplished by adding 10 μL of 10 x reaction buffer and 10 μL of DNase I (1 unit/μL) to an 80 μL RNA sample. After 15 min of incubation at room temperature, the reaction was stopped by the addition of 10 μL of Stop Solution. Next, the sample was heated at 70 °C for 10 min to denature both the DNase I and the RNA. The RNA purity and concentration were measured spectrophotometrically, aliquoted, and stored at –80 °C for further use.

Specimens were collected via surgery or CT-guided puncture. Specimens included granulation tissue and pus. Blood specimens were obtained when all patients were enrolled in this study. The samples then were sent to the laboratory for further processing. Granulation tissue samples were cut into small pieces on a disposable Petri dish support by using a scalpel. Each granulation tissue sample was weighed, and then added with phosphate-buffered saline at the rate of 1 g/ml. The mixture was homogenized with a FastPrep-24 instrument (MP Biomedicals Europe) for 100 s at 6 m/s by using MP Bio FastPrep-24. During homogenization, the tube containing the mixture was removed from the FastPrep-24 instrument every 20 s and cooled on ice for 30 s. Pus samples or the mixtures of homogenized granulation tissue (a 600 μl volume of each) from all patients were each mixed with 1 g of 0.5 mm diameter glass beads and then placed on a vortex mixer for 30 min at 3,000 rpm.

Mtb (strain Erdman) infections were performed via the aerosol route as described previously [13 (link)]. Infections were performed using a Glas-Col (Terre Haute, IN) full body inhalation exposure system. Mice received an inoculation dose of 25–75 CFU/mouse, as measured by plating undiluted lung homogenate within 24 hours of infection. At different times post-infection, mice were euthanized, organs were aseptically removed, individually homogenized in the FastPrep24 (MP Biomedicals, Santa Ana, CA, USA), and viable bacteria were enumerated by plating 10-fold serial dilutions of organ homogenates onto 7H11 agar plates (Hardy Diagnostics, Santa Maria, CA, USA). Mtb (strain H37Rv) in vitro infections of macrophages and DCs were performed as described previously [52 (link)]. H37Rv was grown and prepared as described [53 (link)]. Bacteria were counted in a Petroff-Hausser counter (Hausser Scientific, Horsham, PA, USA) and added to macrophages or DCs at an intended multiplicity of infection (MOI) of 5–10 for three hours. Cultures were washed three times to remove extracellular bacteria, and T cells were added the same day. For CFU measurement, cells were lysed with 1% Triton X-100/PBS and lysate from quadruplicate conditions on d0 and d5 post-infection, and 5-fold dilutions were plated on Middlebrook 7H11 agar plates (Hardy Diagnostics).

Following euthanasia, whole brain tissues were collected within individual Eppendorf tubes containing 1 ml dPBS and cOmplete™ EDTA‐free Protease Inhibitor, prior to storage at −70/80°C or immediately homogenized using a Retsch MM 301 Mixer Mill or FastPrep®‐24 (MP Biomedicals) 2x for 1 min using tungsten beads. Collected blood was spun at 6000 g for 8 min to obtain sera. Cytokine measurements from tissue supernatant and sera were obtained with the BD Biosciences Cytometric Bead Array Mouse Inflammation Kit. Cytokine measurements from whole brain samples were normalized to tissue weight.

The RNAlater-stored brain tissues were homogenized with FastPrep-24 (MP Biomedical, USA). Total DNA was isolated by the DNeasy Blood and tissue kit (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer's protocols with modifications. In brief, 20–30 mg of homogenized brain tissue was lysed and applied to spin column. After the column was washed with AW1 buffer, DNase I (Qiagen) was added to the column and incubated at room temperature for 15 min to remove residual DNA. To completely remove possible contamination of the residual DNA, eluted RNA was further treated with rDNase I at 37 °C for 20 min according to manufacturer's protocol (DNase treatment & removal, Ambion). Quantification of DNA and RNA was done on Nanodrop spectrophotometer (Thermo Scientific).

Genomic DNA was isolated from the 2040 patient samples using FastPrep24 lysis method (MP Biomedicals, California, USA) as per standard protocol and quantified using Qubit (Life Technologies, Carlsbad, California, USA). Libraries for WGS were prepared using Nextera XT DNA Library Prep Kit, and sequencing was performed on the Illumina NextSeq500 machine as per the manufacturer’s protocol (Illumina Inc., San Diego, California, USA) producing 2 × 151 base pair reads.