Transfection procedure

For the LmjF.22.0600 overexpression, 3 × 10⁷ L. major cells in logarithmic phase were transfected with 10 µg of the recombinant vector pTH6nGFPc containing the LmjF.22.0600 sequence, in 100 µl of Human T Cell Nucleofector solution (Lonza, Basel, Switzerland).

For the LmjF.22.0600 deletion, 10⁷ L. major cells expressing Cas9 and T7 (kind gift from Eva Gluenz, University of Oxford) in logarithmic phase were transfected with 30 µg of purified PCR. The transfection buffer was composed of 90 mM sodium phosphate, 5 mM potassium chloride, 0.15 mM calcium chloride, 50 mM HEPES, pH 7.3. The same transfection without purified PCR was used as control.

The transfections were performed in 2 mm gap cuvettes (Lonza) with program X-001 of the Amaxa Nucleofector II (Lonza). Transfected cells were immediately transferred into 5 ml pre-warmed medium and left to recover overnight at 26 °C before adding 30 µg/ml hygromycin B (Thermo Fisher Scientific) for the overexpression and two drugs for the deletion: geneticin (Sigma-Aldrich) at 20 µg/ml and puromycin (Sigma-Aldrich) at 30 µg/ml.