library_construction_protocol: Each embryo was photographed and then minced into fine pieces using a sterile scalpel. The minced embryo was incubated at 37°C with Trypsin (GibcoTM TrypLETM Express Enzyme (1X), phenol red) to dissociate the cells into a single cell suspension. Trypsin treatment was terminated after ten minutes by applying an inhibitor (GibcoTM Defined Trypsin Inhibitor). The suspension was triturated 50 times up and down through a 1ml pipet tip and then passed through a 70μm cell strainer (FlowmiTM Cell Strainer) to remove any remaining clumps. To stain the cells for viability the suspension was incubated for 12 minutes on ice with a Live/Dead stain (Invitrogen LIVE/DEADTM Fixable Near-IR Dead Cell Stain Kit; L34975). Suspended cells were washed once in nuclease-free PBS, fixed in methanol and stored in methanol at -80°C for up to several months. The fixed cell solutions were resuspended in a permeabilization buffer (PB; 2% BSA, 100U/ml RNase-inhibitor, 5mM 1,4-Ditiotreitol, 0.1M MOPS pH 7.5, 1mM EGTA, 2mM MgSO4, 125mM NaCl and 0.1% Saponin), and incubated on ice for 5min. Once permeabilized, the cells were stained with a mix of DAPI (1:1000; ThermoScientific 62248), which stains DNA, and a FITC-conjugated HNK-1 antibody (1:100; Anti-Hu CD57 eBioscience 11-057742) in MRDB (a buffer identical to PB but lacking saponin), followed by a washing step with the same buffer and passed through a 70μm cell strainer. Dissociated cells were sorted in a FACS ARIA III machine. Debris, doublets and cells that were dead before fixation were removed by three gates. The first gate excluded debris by excluding cells with low values in both forward and side scatter area. The second gate removed doublets by excluding cells with high values in forward scatter width, which can be clearly made out when forward scatter width and height are plotted against each other. The third gate further removed additional (large) debris, non-nucleated cells and cells that were dead before fixation by only selecting cells with high DAPI fluorescence and low Live/Dead fluorescence. The remaining cells were inspected and sorted based on their anti-HNK-1 (FITC) signal. The unsorted and leniently sorted samples were processed into cDNA using 10X Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index). The strictly sorted sample was processed to cDNA following the Smart-seq3 protocol.