Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool to explore cells. With an increasing number of scRNA-seq data analysis tools that have been developed, it is challenging for users to choose and compare their performance. Here, we present an overview of the workflow for computational analysis of scRNA-seq data. We detail the steps of a typical scRNA-seq analysis, including experimental design, pre-processing and quality control, feature selection, dimensionality reduction, cell clustering and annotation, and downstream analysis including batch correction, trajectory inference and cell–cell communication. We provide guidelines according to our best practice. This review will be helpful for the experimentalists interested in analyzing their data, and will aid the users seeking to update their analysis pipelines.