Pregnant C57BL/6 mouse females were purchased from Charles River and delivered one day before or on the day of embryo harvest. Mouse embryos were dissected at time-points E6.5, E6.75. E7.0, E7.25, E7.5, E7.75, E8.0, E8.25 and E8.5. As previously reported6 (link), development can proceed at different speeds between embryos, even within the same litter (Fig. 1a; Extended Data Fig. 1). Consequently, we adopted careful staging by morphology (Downs and Davies staging6 (link)) to exclude clear outliers. Following euthanasia of the females using cervical dislocation, the uteri were collected into PBS with 2% heat-inactivated FCS and the embryos were immediately dissected and processed for scRNA-seq. Two samples contained pooled embryos staged across several time-points. Cells from these samples are denoted as “Mixed” in Figures, and “mixed_gastrulation” in Supplementary Information Table 4. Embryos from the same stage were pooled to make individual 10X samples, and single-cell suspensions were prepared by incubating the embryos with TrypLE Express dissociation reagent (Life Technologies) at 37 °C for 7 min and quenching with heat inactivated serum. The resulting single-cell suspension was washed and resuspended in PBS with 0.4% BSA, and filtered through a Flowmi Tip Strainer with 40 µm porosity (ThermoFisher Scientific, #136800040). Cell counts were then assessed with a haemocytometer. scRNA-seq libraries were subsequently generated using the 10X Genomics Chromium system (version 1 chemistry) and samples were sequenced according to the manufacturer’s instructions on an Illumina HiSeq 2500 platform. Supplementary Information Table 1 contains detailed information on embryo collection, and Supplementary Information Table 4 contains metadata for each sequenced cell. Sample sizes were chosen to maximise the number of recovered cells from each experiment and to obtain total cell numbers similar to the estimated cell numbers in mouse embryos at their respective stages. The sample sizes were also dependent on the number of viable embryos from each litter. Cells were partitioned to prevent overloading of a single 10X lane.