Differentiation and single-cell RNA-seq analyses of human pluripotent-stem-cell-derived renal organoids

Abstract

Summary: Here, we present a protocol for the maintenance and differentiation of human pluripotent stem cells into renal organoids. We describe steps for using a series of readily made differentiation media, multiplexed sample single-cell RNA-seq analysis, quality control, and validation of organoids using immunofluorescence. This provides a rapid and reproducible model of human kidney development and renal disease modeling. Finally, we detail genome engineering using CRISPR-Cas9 homology-directed repair for the generation of renal disease models.For complete details on the use and execution of this protocol, please refer to Pietrobon et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Sequence Analysis
• Cell Biology
• Cell Culture
• Single Cell
• Genomics
• RNA-seq