Transgenic T. brucei brucei Parasite Culture

All transgenic T. bruceibrucei parasites used in this study were derived from monomorphic T. bruceibrucei 2T1 bloodstream forms (63 (link)) and were cultured in HMI-11 (HMI-9 [GIBCO] containing 10%, vol/vol, fetal bovine serum [GIBCO], Pen-Strep solution [penicillin at 20 U ml⁻¹, streptomycin at 20 mg ml⁻¹]) at 37°C and 5% CO₂ in vented flasks. Selective antibiotics were used: 5 μg ml⁻¹ blasticidin or hygromycin and 2.5 μg ml⁻¹ phleomycin or G418. RNAi was induced in vitro with tetracycline (Sigma-Aldrich) in 70% ethanol at 1 μg ml⁻¹. The endogenous Ty, mNeonGreen experiment was performed using the pPOTv6 vector (64 (link)). The generation of inducible TbCLK1 and KKT2 RNAi was done as previously described (20 (link)).