Figure S1.

Stage-Specific RNA-Seq and Forward-Genetic Screening in a Differentiation Reporter Strain, Related to Figure 1

(A) Sample FACS plots for the bradyzoite reporter strain at 24 or 48 h of growth under unstressed or stressed conditions. 10,000 events per plot. Gates for mNG⁺ and mNG^– populations are highlighted.

(B) Principal component analysis of stage-specific RNA-seq replicates.

(C–D) Comparison of differentially expressed genes identified in this study and a previously published dataset (Fritz et al., 2012). Color assigned by adjusted p-value < 0.001 (C) or rank of base mean expression as calculated by DESeq2 (D).

(E) Efficiency of construct integration into the bradyzoite reporter strain. Plaquing efficiency was compared between parasites transfected and selected for integration of a plasmid encoding a gRNA against SAG1 and the pre-transfection population to obtain a viability-normalized integration rate. Mean ± SD is plotted for n = 3 independent experiments.

(F) Generation of a BFD1-deficient reporter strain by introducing a frameshift using Cas9. The bradyzoite reporter strain was transfected with a plasmid encoding a gRNA targeting the first exon of TGME49_200385 (BFD1) to isolate a strain with a frameshift mutation (BFD1^frameshift), resulting in a premature stop codon after amino acid 251.