Preparation of Cells from Embryonic Organs for Single-Cell RNA Sequencing

Although single-cell RNA sequencing (scRNA-seq) has become one of the most powerful methods available for transcriptome analysis, the quality of scRNA-seq data largely depends on cell preparation. Cell preparation from cultured cells and tissues requires different methods because of the inherent differences between these two categories of cells. Compared to cultured cells, tissues have more extracellular matrix, and the cells are generally more adherent and thus difficult to dissociate. The challenge is to achieve sufficient dissociation, cell counts, and viability all at the same time. This protocol describes approaches that help achieve these goals. These include a cold dissociation technique using cryophilic proteases active at cold temperature, timing of trituration during protease digestion, as well as filtration and washing methods that optimize cell viability and retention. Materials and equipment that optimize the process will also be discussed. © 2019 by John Wiley & Sons, Inc.