Figure EV1. Schematic representing the cellular barcoding strategy.

1. Briefly, a double‐stranded oligonucleotide is synthesized bearing a semi‐random sequence formed by the alternation of weak (W = A or T) and strong bases (S = G or C) flanked by two fixed sequences that serve as primer binding sites for PCR amplification. This pool of DNA molecules is cloned in a vector that has homology sequences which promote the integration of the vector into the 18s rRNA locus in Leishmania genome.
2. After transfection, barcoded parasites are selected with hygromycin, as the barcoding vector also has a hygromycin‐resistance gene. The eGFP gene allow to monitor by flow cytometry the potential presence of remaining non‐barcoded cells after hygromycin selection.