Proteinase k

The frozen, fixed cells (~ 2 million) were incubated in a 37 °C water bath until thawed (~ 1 min). After thawing, the cells stored in 1 ml freezing buffer were diluted with 3 ml of PBSB. The fixed cells were collected by centrifuging at 500 r.c.f for 10 min at 4 °C, resuspended again in 1 ml PBSB, and then centrifuged at 500 r.c.f for 5 min at 4 °C to wash the cells. Following washing, the nuclei were isolated and tagmented as described in the ATAC-seq protocol for native samples described above. After tagmentation, crosslinks were then reversed for the fixed, tagmented nuclei by adding 60 μl of reverse-crosslinking buffer containing 0.067% SDS and 1.33 mg/ml Proteinase K (Qiagen, cat. no. 19133) in Qiagen Buffer EB (final concentration of 0.05% SDS and 1 mg/ml Proteinase K) directly to the tagmentation reactions, and incubating the samples at 65 °C for 15 h in a thermomixer with shaking at 1000 r.p.m. Following crosslink reversal, tagmented DNA was purified, amplified and size-selected as described in the ATAC-seq protocol for native samples.

Tachyzoite numbers were estimated via a quantitative PCR according to Cortes, et al.^{69 (link)}. Therefore, biological triplicates with technical duplicates were processed. Firstly, cell culture supernatants containing free-released tachyzoites (=extracellular tachyzoites) were collected at 48 h p. i. and pelleted (600 × g, 15 min). In addition, the remaining host cells carrying not yet released tachyzoites (=intracellular tachyzoites) were trypsinized and pelleted (600 × g, 15 min). All cell/parasite pellets were treated with 200 μL of cell lysis buffer containing 0.32 M sucrose, 1% Triton X-100, 0.01 M Tris-HCl (pH 7.5), 5 mM MgCl₂ and incubated in 100 μL 1X PCR buffer (Quanta) containing 20 μL proteinase K (20 mg/mL; Qiagen) at 56 °C for 1 h. proteinase K was heat-inactivated by heating the samples (95 °C, 10 min) and the DNA-containing samples were frozen at −20 °C until further use. Real-time PCR was performed in a total volume of 20 μL containing 2 μL DNA of test samples, 400 nM of each primer, 200 nM probe and 10 μL PerfeCTa MasterMix (Quanta) at the following cycling conditions: 95 °C for 10 min; 40 cycles at 95 °C for 10 s, 60 °C for 15 s and 72 °C for 30 s.

Community DNA was extracted using the Qiagen Plant Minikit (Qiagen) and partially automated using a QIAcube (Qiagen) with modifications as described below. The filters were thawed at room temperature and 400 μl AP1 buffer was added to the sample tubes. Thereafter the tubes were subjected to three freeze–thaw cycles using liquid nitrogen and a 65°C water bath (30s and 2 min respectively) followed by bead‐beating (FastPrep‐24 bead beater; MP Biomedicals, Irvine, CA, USA) at full speed for 2 min. The samples were Proteinase K treated for 1 h at 55°C with moderate shaking using 45 μl of Proteinase K (Qiagen, 20 mg ml⁻¹) and then treated with 4 μl RNase A (Qiagen, 100 mg ml⁻¹) and incubated for 10 min at 65°C. The filters were removed using sterile needles, 130 μl AP2 buffer was added to each tube, and samples were then vortexed and incubated on ice for 10 min. To pellet the precipitates and beads the tubes were centrifuged for 5 min at 18 800g at 4°C, in a Sorvall Legend Micro 21 (Thermo Electron Corporation) centrifuge and the supernatant was transferred to 2 ml sample tubes and placed in the QIAcube for further extraction steps according to the manufacturer's protocol. The samples were eluted using 100 μl AE buffer and concentrations were measured fluorometrically using Picogreen (ThermoFisher).

To test the addition of proteinase K to the lysis buffer, each detergent lysis buffer was supplemented with 0.1 AU/mL proteinase K (Qiagen, Hilden, Germany) and 0.83 mM final concentration EDTA (Sigma, Steinheim, Germany) to prevent heat damage to RNA during heat inactivation. After, mixed 1:1 with VPM or vRNA in nuclease-free water (NF-H₂O) samples were incubated in thin-walled tubes in a PCR machine for 30 min at 56 °C prior to heat inactivation for 10 min at 95 °C. The inactivated lysate was added at 10% final volume to the RT-qPCR reaction; this was set up and analyzed as described before.