Approaches for single-cell RNA sequencing across tissues and cell types

Single-cell sequencing of RNA (scRNA-seq) has advanced our understanding of cellular heterogeneity and signaling in developmental biology and disease. A large number of complementary assays have been developed to profile transcriptomes of individual cells, also in combination with other readouts, such as chromatin accessibility or antibody-based analysis of protein surface markers. As scRNA-seq technologies are advancing fast, it is challenging to establish robust workflows and up-to-date protocols that are best suited to address the large range of research questions. Here, we review scRNA-seq techniques from mRNA end-counting to total RNA in relation to their specific features and outline the necessary sample preparation steps and quality control measures. Based on our experience in dealing with the continuously growing portfolio from the perspective of a central single-cell facility, we aim to provide guidance on how workflows can be best automatized and share our experience in coping with the continuous expansion of scRNA-seq techniques.