2.4. DNA extraction

Lymph nodes were thawed on ice for 30 min before processing. All work was performed in sterile conditions in a Class II biosafety cabinet, using aseptic techniques and DNase-free instruments and consumables. Excess Allprotect Tissue Reagent was removed, and nodes were washed in 1 mL sterile phosphate-buffered saline (PBS; pH 7.4 [137 mM NaCl; 2.68 mM KCl; 9.94 mM Na₂HPO₄; 1.76 mM KH₂PO₄]). MLNs were weighed, and samples of ~25 mg were used for DNA extraction. Total DNA was extracted from samples using a QIAamp cador pathogen Mini Kit [Qiagen, UK], using the manufacturer’s instructions. In brief, tissue lysis buffer [180 µL; Buffer ATL] was added to the node tissue and the sample was homogenized for 1 min using a motorized pestle. Proteinase K enzyme [20 µL] was added to this to commence enzymatic tissue digestion. Samples were incubated overnight at 56°C with constant shaking. Following this, digested tissue [200 µL] mixed with sterile PBS [200 µL] was transferred to Pathogen Lysis Tubes S [Qiagen, UK], pre-prepared with tissue lysis buffer [Buffer ATL] and anti-foaming reagent [Reagent DX] [100 µL total] for mechanical disruption of hard-to-lyse bacteria by glass beads. This was completed by vigorous shaking for 10 min. Buffer VXL [100 µL], which ensures the inactivation of nucleases when in the presence of proteinase [100 µL], was added to the pre-treated samples [enzymatically digested and mechanically disrupted], mixed well, and left to incubate at room temperature for 15 min. Binding reagent [Buffer ACB; 350 µL] was added to samples, mixed well, and transferred to spin columns. DNA was then isolated following a series of centrifugations using QIAamp spin columns and buffer solutions. DNA was eluted in 100 µL elution buffer [Buffer AVE] [2 × 10-min elutions], and quantity and quality were assessed using the 260/280 function on the Spectrostar Nano plate reader LVis plate function [BMG Labtech]. Extracted DNA was stored at –20°C for subsequent polymerase chain reaction [PCR].