An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis

Abstract

Summary: Here, we describe a protocol for single-cell isolation from the primary culture of normal human epidermal keratinocytes derived from neonatal foreskin. The cell culture conditions have been optimized for inducing expression of keratinocyte differentiation markers. Cells are cultured in the absence or presence of a bioactive lipid lysophosphatidic acid (LPA). Single cells are isolated by Fluidigm C1 system. This is followed by cDNA library preparation using Takara SMART-Seq v4 Ultra and Illumina Nextera XT kit for RNA sequencing.For complete details on the use and execution of this protocol, please refer to Siriwach et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Sequence analysis
• Cell Biology
• Single Cell
• Developmental biology
• RNAseq