Single-cell sequencing measures the sequence information from individual cells using optimized single-cell isolation protocols and next-generation sequencing technologies. Recent advancement in single-cell sequencing has transformed biomedical research, providing insights into diverse biological processes such as mammalian development, immune system function, cellular diversity and heterogeneity, and disease pathogenesis. In this article, we introduce and describe popular commercial platforms for single-cell RNA sequencing, general workflow for data analysis, repositories and databases, and applications for these approaches in biomedical research. © 2020 American Physiological Society. Compr Physiol 10:767-783, 2020.