T. brucei cell culture and transfection

The T. brucei Lister 427 (29–13) strain that carries the inducibly-expressed RBP6 (Tb927.3.2930) transgene at the rDNA spacer (pLew100.v5-BSD) was cultured at 28 °C and 5% CO₂ in Cunningham’s media supplemented with 10% Tet-system approved heat-inactivated Fetal Bovine Serum (FBS) and 2 mM l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 15 μg/ml G418, 50 μg/ml hygromycin B, and 10 μg/ml blasticidin. A total of 1 × 10⁸ of the RBP6 overexpression cells were used to transfect an inducible hairpin RNAi construct. Cells were centrifuged at 3500 RPM for 6 min, washed in Cytomix (20 mM KCl, 0.15 mM CaCl₂, 10 mM K₂HPO₄, 25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes), 2 mM ethylenediaminetetraacetic acid (EDTA) and 5 mM MgCl₂, pH7.6), and resuspended in 500 μl Cytomix. Subsequently, 25 μg of linearized plasmid DNA was mixed with the solution and cells were pulsed twice at 1600 V with a time constant of 0.6 ms using a GenePulser Xcell (BioRad, Hercules, CA, USA). The selective drug, phleomycin, was added to the culture medium 24 h after electroporation at a final concentration of 2.5 μg/ml.