For dissociation, floating EBs (on day8) / attached EBs (on day16) were washed with DPBS and incubated with Accumax (Sigma-Aldrich, SCR006) for 10-20 min at 37°C. Next, EBs were pipetted up and down with a p1000 pipette until they were completely dissociated to single cells and DFK20 medium was added. Cells were pelleted at 300 xg for 5 min and then resuspended in 1 mL DPBS. If remaining cell clumps were observed, the cell suspension was filtered through a 40 µm cell strainer, then cells were counted using a Countess II automated cell counter (Thermo Fisher Scientific, C10228). Equal cell numbers from each cell line were pooled, washed with DPBS + 0.04% BSA and resuspended in an appropriate volume of DPBS + 0.04 % BSA leading to a final concentration of 800 - 1000 cells/µL. The pooled cells were kept on ice and loaded on a 10x Chromium Controller Chip within 15 min. scRNA-seq libraries were generated using the 10x Genomics Chromium Next GEM Single Cell 3´Kit V3.1 workflow in three replicates. Each time, evenly pooled single cells from the different clones were loaded on 2 to 6 lanes of a 10x chip, with a targeted cell recovery of 16,000 cells per lane.