AbstractDuring the last decade, high‐throughput sequencing methods have revolutionized the entire field of biology. The opportunity to study entire transcriptomes in great detail using RNA sequencing (RNA‐seq) has fueled many important discoveries and is now a routine method in biomedical research. However, RNA‐seq is typically performed in “bulk,” and the data represent an average of gene expression patterns across thousands to millions of cells; this might obscure biologically relevant differences between cells. Single‐cell RNA‐seq (scRNA‐seq) represents an approach to overcome this problem. By isolating single cells, capturing their transcripts, and generating sequencing libraries in which the transcripts are mapped to individual cells, scRNA‐seq allows assessment of fundamental biological properties of cell populations and biological systems at unprecedented resolution. Here, we present the most common scRNA‐seq protocols in use today and the basics of data analysis and discuss factors that are important to consider before planning and designing an scRNA‐seq project. © 2018 by John Wiley & Sons, Inc.