 Abstract RNA sequencing, RNA-SEC, is a genomic approach for detecting and quantifying messenger RNA molecules in biological samples, useful for studying cellular responses. However, it has hindered direct assessment of the fundamental unit of biology, the cell. Single cell RNA sequencing, SCRNA-SEC, was developed to address this issue by analyzing individual cells. While initially conducted by specialist laboratories, SCRNA-SEC is now commercially available and can be used by any biomedical researcher or clinician to make discoveries. This review provides a practical guide for designing SCRNA-SEC studies, including experimental hardware, protocol choice, quality control, data analysis and biological interpretation. This article was authored by Ashraf Alhuk, Jessica Engel, Sarah A. Teachman, and others.