Early mouse embryos were isolated at E5.5 (from pregnant CD1 females, purchased from Charles River, UK). Following dissection from the decidua, embryos were cultured overnight in N2B27 “poor” media (same formulation as N2B27 media but supplemented with 0.5xB27 supplement and 0.5xN2 supplement) with pan-caspase inhibitors (100 μM, Z-VAD-FMK, FMK001, R&D Systems, USA) or equal volume of vehicle (DMSO) as control. On the next morning, embryos were processed for single cell RNA-Seq (scRNA-seq) or functional validation (Δψm analysis and immunohistochemistry for markers of loser cells).
For the scRNA-seq and Δψm analysis embryos were dissociated into singe-cells. Briefly, up to 12 embryos were dissociated in 600 μL Acccutase (A6964, Sigma, UK) during 12 min at 37°C, tapping the tube every two minutes. Accutase was then neutralised with equal volume of FCS, cells span down and stained with TMRM, for Δψm analysis, or directly re-suspended in 300 μL DPBS with 1% FCS, for single cell sorting and RNA-seq. Sytox blue (1:1000, S34857, Thermo Fisher Scientific, UK), was used as viability staining.
For the scRNA-seq and Δψm analysis embryos were dissociated into singe-cells. Briefly, up to 12 embryos were dissociated in 600 μL Acccutase (A6964, Sigma, UK) during 12 min at 37°C, tapping the tube every two minutes. Accutase was then neutralised with equal volume of FCS, cells span down and stained with TMRM, for Δψm analysis, or directly re-suspended in 300 μL DPBS with 1% FCS, for single cell sorting and RNA-seq. Sytox blue (1:1000, S34857, Thermo Fisher Scientific, UK), was used as viability staining.