Live Droplet Single-Cell RNA Sequencing

Abstract

Single-cell RNA sequencing has emerged as one of the most influential methods of the past decade, enabling the dissection of cellular heterogeneity and the discovery of rare cell types. However, most workflows require cell lysis and provide only terminal snapshots, limiting their ability to capture dynamic cellular trajectories and temporal variations within the same cell. Herein, we present Live Droplet single-cell RNA sequencing (Live D-seq), a technology that enables scalable transcriptome-wide profiling of individual living cells over time. Live D-seq combines longitudinal intracellular sampling of live, barcoded cells with particle-templated emulsification and barcoded hydrogel beads, enabling repeated samples from the same cell lineage to be linked across time points. We showcase Live D-seq's capabilities by (i) demonstrating minimal cross-contamination in cross-species experiments, (ii) precisely mapping cellular barcodes to hydrogel bead barcodes, and (iii) tracking epithelial-to-mesenchymal transition dynamics in A549 cells over time. Altogether, Live D-seq enables repeated, non-destructive measurements of gene expression in the same single cells, offering a framework to study longitudinal single-cell biology in health and disease.