library_construction_protocol: Gastruloids were collected, pooled in a 1.7 ml Eppendorf tube and washed twice in 1 ml of PBS. The number of gastruloids used in each condition was chosen to ensure that more than 300 000 cells were obtained. They were then dissociated in 100 µl of Accutase (Stempro) for 5 min at 37°C. Full dissociation was completed using mechanical dissociation and verified to ensure the absence of doublet cells. Cells were then resuspended in 200 µl of PBS and counted on a Countess 3 automated cell counter (Invitrogen). All centrifugations were done at 400g for 5 minutes. Samples were then analysed with the standard 10x genomics pipeline (Chemistry v3). After counting, dissociated cells were diluted in PBS BSA 0.04% (Millipore, A1595) to 1x106 cells/ml. Samples were then filtered using a 40 µm cell strainer (Flowmi, BAH136800040). The pool of cells was counted and subjected to single cell RNA-seq following manufacturer's recommendation, we aimed for a recovery of c.a. 6000 cells per sample. cDNA preparations were performed according to 10x Genomics recommendations, amplified for 10–12 cycles and verified on fragment analyser.