GSM9131352: eDS072_TGGT1_278440_Rep1; Toxoplasma gondii; OTHER
Intracellular parasites were harvested by mechanical disruption through a 27-gauge needle and filtration through a 5  μm membrane. A total of ~5 x 10⁷ parasites were pelleted (1500 x g, 10  min) and resuspended in wash buffer (20 mM HEPES-NaOH pH 7.5, 150  mM NaCl, 0.5  mM Spermidine, 1x EDTA-free Protease Inhibitor Cocktail) and attached to 100 μl BioMag Plus Concanavalin A beads (Polysciences) activated in bead activation buffer (20 mM HEPES-KOH pH 7.9, 10  mM KCl, 0.1  mM CaCl2, 0.1 mM MnCl2). Parasites were bound to the beads by incubation for 15 min at room temperature and rotation. Bead-bound parasites were blocked with wash buffer supplemented with 2 mM EDTA for 5 min at room temperature followed by binding of 1 µg NbALFA-MNase per sample in ice-cold wash buffer supplemented with 0.025 % Digitonin (Invitrogen) for 1 hr at 4 °C. After NbALFA-MNase binding, bead-bound parasites were washed three times with ice-cold wash buffer supplemented with 0.025 % Digitonin for 5 min at 4 °C. The cutting reaction was started by the addition of ice-cold wash buffer supplemented with 0.025 % Digitonin and 2 mM CaCl2 and incubation for 60 min on ice. The reaction was stopped by the addition of 4xSTOP buffer (400 mM NaCl, 40 mM EDTA, 8 mM EGTA, 100 µg/ml RNaseA, 80 µg/ml Glycogen and 0.025 % Digitonin) to 1x and incubation at 37 °C for 20 min. The DNA was extracted using a modified organic extraction and ethanol precipitation protocol. CUT&RUN libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB) according to the manufacturer's instructions with 11–12 PCR amplification cycles. Size selection and clean-up steps were performed with AMPure XP beads (Beckman Coulter).