the region (Table S1). Suspensions were then passed through a pre-wet 40 μm filter into a new tube on ice. N=2 10 μl samples were then drawn from the tube and mixed 1:1 with 10 μl of 2× dye mix containing Dissociation Buffer and 20 μM EthD-1 (Thermo Fisher Scientific, L-3224), 20 μM Calcein-AM (Thermo Fisher Scientific, L-3224) and 40 μM Hoechst 33342 (Thermo Fisher Scientific, 62249). After 5 minutes incubation, 10 μl from each sample was loaded onto a haemocytometer (Propper, 090001) and imaged using a fluorescent microscope (Zeiss, Axio Observer Z1). For each of the two samples, three random locations were imaged using DIC and three fluorescent channels to capture the dyes. These images were used to calculate cell concentrations and metrics of cell intactness. Drop-seq analysis was performed on 44 cell suspensions, with 3–7 replicates per region.
