Dnase 1 reaction buffer

For the HCT116 cell positive control, 5 × 10⁴ cells were suspended in 100 μL PBS for each treatment group. For conditioned media and plasma samples, 100 μL of sample for each treatment group was pretreated with 1 μL Triton X-100 (Thermo Fisher Scientific) or PBS and incubated for 10 minutes at room temperature. Next, samples were treated with 2 U DNase I (New England Biolabs) or PBS, along with 1× DNase I Reaction Buffer (10 mM Tris-HCl, 2.5 mM MgCl₂, 0.5 mM CaCl₂, pH 7.6; New England Biolabs) and incubated for 30 minutes at 37°C. The DNase I reaction was halted by adding 1 μL of 0.5 M EDTA and incubating for 10 minutes at 75°C. For plasma samples, the heat inactivation step was omitted (as heating increased plasma viscosity, making efficient DNA-IP difficult), and 3 μL of 0.5 M EDTA was added instead to halt DNase I activity. Treated samples were then subjected to IP and DNA purification as described above.

Five-hundred microliters of each fraction were used for nucleic acid extraction twice with phenol/chloroform/isoamyl alcohol (25:24:1), pH 8.0 (Thermo Fisher Scientific) and twice with chloroform. The aqueous phase was transferred to a new tube, and the nucleic acids were precipitated with 300 mM sodium acetate, pH 5.2 and 2.5 equivalent volume of absolute ethanol. After chilling for 1 h at −86 °C, precipitated nucleic acids were pelleted by centrifugation (19,000× g, 20 min, 4 °C), and the pellets were resuspended in 100 µL water. Eighteen microliters of each nucleic acid solution was mixed with 2 µL of 10× DNase I reaction buffer (New England Biolabs, Ipswich, MA, USA), to which the vehicle PBS, 2.5 units RNase A and 100 units RNase T1 (RNAse cocktail A+T1, Invitrogen), 1 unit DNase I (New England Biolabs), or RNase and DNase together were added, and the tubes were incubated for 1 h at 37 °C. After 1 h, 20 µL stop solution (50% formamide, 50 mM EDTA, 0.1% bromophenol, and 0.1% xylene cyanol) was added, and samples were subjected to a denaturing 8 M urea PAGE. The gel was run at a 1000 V constant for 5 h and then stained with Sybr^® Gold stain (Thermo Fisher Scientific) for 20 min and visualized by UV at 254 nm.

For DNA extraction from standard bacterial strains, a single colony (with the exception of P. mirabilis CMCC 49005 due to its swarming growth on blood agar) from each plate was added to 100 µL of QuickExtract DNA Extraction Solution (Epicentre). The samples were incubated at 65 °C for 15 min and 98 °C for 2 min. The extracted genomic DNA (gDNA) samples were stored at −80 °C until use. A 4‐fold dilution series of a contrived sample containing eight different types of bacterial gDNA was prepared in 1 mg·mL⁻¹ BSA (Sigma).
For DNA extraction from clinical samples, an aliquot of remnant sample (2 mL) was pelleted at 8000 g for 10 min. The bacterial pellet was then resuspended in a 100‐µL solution containing 1 µL of DNase I (New England Biolabs) and 10 µL of DNase I Reaction Buffer (New England Biolabs). After incubation at 37 °C for 10 min, the sample was pelleted at 8000 g for 10 min. This DNase treatment was used to degrade extracellular DNA present in the samples to prevent the overestimation of bacterial density. After discarding the supernatant, an aliquot of QuickExtract DNA Extraction Solution (100 µL for BALF and 200 µL for urine) was added to the sample. DNA extraction was performed as described above. This DNA extraction process resulted in a 20‐fold enrichment in bacterial gDNA concentrations for BALF samples and a 10‐fold enrichment for urine samples.