Stock solutions of barcoding dyes CBD 450 (BD Biosciences), CBD 500 (BD Biosciences), and DL800 (Thermo Fisher Scientific) were prepared as per the manufacturer’s instructions in DMSO in polypropylene 96-well plates and stored at −80°C. Different combinations of these dyes were used to produce a different number of barcoded cell populations specific to each study as follows. For the KP study, 80 populations were resolved using final concentrations of CBD 450 (0.000, 0.015, 0.075, and 0.300 mg/ml) combined with CBD 500 (0.000, 0.038, 0.188, and 0.750 mg/ml) and DL800 (0.000, 0.011, 0.033, 0.100, and 0.300 μg/ml; figs. S1 and S2). For the TI study, 64 barcoded populations were resolved using final concentrations of CBD 450 (0.000, 0.015, 0.050, and 0.150 mg/ml) combined with CBD 500 (0.000, 0.038, 0.125, and 0.375 mg/ml) and DL800 (0.000, 0.017, 0.050, and 0.150 μg/ml; fig. S10). For the DR study, four populations were resolved using final concentrations of DL800 (0.000, 0.017, 0.050, and 0.150 μg/ml; fig. S16). Cells in the CV study were not barcoded and treated with the vehicle (PBS) instead.

Fixed cells were washed with PBS and permeabilized in 100 μl of ice-cold methanol (Fisher Chemical) for 20 min at 2°C using a Biomek NX liquid handler to allow penetration of antibodies against intracellular epitopes. The barcoding dyes were diluted in ice-cold PBS and 100 μl per well added to the suspension of cells in methanol. The final concentration of DMSO from the barcoding dyes at this stage was 3.5%. The barcoding reaction was incubated in the dark for 30 min at 2°C, and the cells were washed five (KP and TI) or four times (DR and CV) in ice-cold fluorescence-activated cell sorting (FACS) buffer [PBS with 0.5% bovine serum albumin (Sigma-Aldrich)]. The barcoding wells were pooled, washed, and resuspended at 1 × 106 cells/ml in FACS buffer for staining.

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