With organoid barcodes we can quantify baseline inter-EB heterogeneity and perturbation-specific changes in cell type composition. We can also apply replicate-based DE analyses, avoiding the high false positive rate caused by inter-EB heterogeneity in standard single cell DE.

Our solution: monoclonal embryoids with an ‘organoid barcode’. This allows us to determine not only the transcriptome and perturbation identity for each cell but also the individual EB of origin - while still growing EBs in a pool, for scalability and shared culture conditions.

Many gRNAs seemed to have significant effects on cell type composition in mosaic EBs, but these were not reproducible across replicates: Stochastic clonal skewing of individual gRNAs in EBs, combined with inter-EB heterogeneity, overwhelms most perturbation effects and reduces statistical power.