Isolation of human ESC-derived cardiac derivatives and embryonic heart cells for population and single-cell RNA-seq analysis

STAR Protoc. 2021 Feb 11;2(1):100339. doi: 10.1016/j.xpro.2021.100339. eCollection 2021 Mar 19.

Abstract

The combination of population and single-cell RNA sequencing analysis using human embryonic stem cell (hESC) differentiation and developmental tissues is a powerful approach to elucidate an organ-specific cellular and molecular atlas in human embryogenesis. This protocol describes (1) cardiac-directed differentiation and isolation of hESC-derived cardiac derivatives with fluorescence-activated cell sorting, (2) isolation of human embryonic heart-derived single cardiac cells, and (3) construction of cDNA libraries with Smart-seq2. These allow for the preparation of human developmental samples for comprehensive transcriptional analysis. For complete details on the use and execution of this protocol, please refer to Sahara et al. (2019).

Keywords: Cell differentiation; Cell isolation; Flow cytometry/mass cytometry; RNA-seq; Single cell; Stem cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation*
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / metabolism
  • Flow Cytometry*
  • Human Embryonic Stem Cells* / cytology
  • Human Embryonic Stem Cells* / metabolism
  • Humans
  • Myocardium* / cytology
  • Myocardium* / metabolism
  • RNA-Seq*
  • Single-Cell Analysis*