A Modified SMART-Seq Method for Single-Cell Transcriptomic Analysis of Embryoid Body Differentiation

Methods Mol Biol. 2021 Oct 19. doi: 10.1007/7651_2021_435. Online ahead of print.

Abstract

Embryoid bodies (EBs) are aggregate of cells that contain three embryonic germ layers. They can be formed by direct differentiation from pluripotent embryonic stem cells (ESCs), which serves as a useful model for understanding early embryo development. Due to the mixture of different cell types, it is necessary to investigate EBs at the single-cell level. Here, we describe a robust and straightforward method for single-cell gene expression profiling during mouse EB differentiation from mouse ESCs (mESCs). The protocol is modified from a widely used method in the SMART-seq family, which only requires standard molecular biology techniques and lab equipment. It allows for accurate 3' counting of transcript at the single-cell level, which helps reveal cellular identities during EB formation. Combined with perturbation experiments, the method provides an opportunity for mechanistic studies of embryo development at the single-cell level.

Keywords: Embryoid body; Genomics; Mouse embryonic stem cells; Single-cell RNA-seq.