Droplet-based single-cell RNA sequencing: decoding cellular heterogeneity for breakthroughs in cancer, reproduction, and beyond

Droplet-based single-cell RNA sequencing (scRNA-seq) has redefined biological research by resolving cellular heterogeneity with an unprecedented precision. This review synthesizes the technological evolution of the 10× Genomics Chromium platform, its Gel Bead-in-Emulsion (GEM)s technology, and end-to-end workflows, while critically addressing persistent challenges, such as cell capture variability (30-75% efficiency), barcode collisions (< 5% multiplet rates), and mRNA capture limitations (10-50% efficiency). We spotlight two transformative applications: (1) cancer research, where scRNA-seq unveils rare circulating tumor cells (CTCs) and dynamic tumor microenvironments, and (2) reproductive medicine, enabling breakthroughs in primordial germ cell (PGC) epigenetics and pre-implantation diagnostics. Unlike prior reviews, we systematically evaluated innovative solutions-including UMIs, computational demultiplexing, and microfluidic cost-reduction strategies (40-60% savings)-and propose a roadmap for integrating spatial transcriptomics and multi-omics approaches. Looking ahead, we identify three frontiers poised to redefine the field: (i) single-cell epigenome-transcriptome co-profiling, (ii) AI-driven analysis of multimodal datasets, and (iii) scalable microfluidics for clinical adoption. This review is of key significance in bridging cutting-edge technology with translational impact in oncology and reproductive biology, while outlining a roadmap for the evolution of the field.