a, Standard droplet-based scRNA-seq, where cells are loaded at a low concentration (limiting dilution) to avoid cell doublets, and most droplets do not receive a cell. b, scifi-RNA-seq, which uses preindexing and droplet overloading to boost the throughput of droplet-based scRNA-seq. c, Detailed method design of scifi-RNA-seq. d, Representative images of droplets containing between one and ten nuclei, showing the overloading of a standard microfluidic droplet generator (10x Genomics Chromium). e, Droplet overloading boosts the percentage of droplets filled with nuclei from 16.4% (obtained for the maximum loading concentration of the standard Chromium protocol) to 95.5% (obtained for 100-fold overloading using 1.53 million nuclei per channel). f, Droplet overloading causes the average number of nuclei per droplet to increase in a controlled fashion while maintaining the desired Poisson-like loading distribution. g, Expected collision rate as a function of the cell/nuclei loading concentration per channel for standard droplet-based scRNA-seq and for scifi-RNA-seq with different numbers of round1 barcodes. h, Due to the high number of microfluidic (round2) barcodes, scifi-RNA-seq exceeds the barcoding capability of three-round combinatorial indexing protocols.