840 Early and late matrix progenitors form the terminally differentiated cell layers of the hair follicle

Abstract

Single cell RNA sequencing (scRNA-seq) has been recently developed to study the gene expression profile of individual cells. This high-resolution transcriptomic analysis is enabling deep insights into biological systems and disease mechanisms. Multiple techniques have been used to capture single cells, including microfluidics platforms, microtiter plates, and droplet-based technologies. These are followed by cell lysis and reverse-transcription (RT) of RNA from the individual cells. Barcoded and multiplexed cDNA are then subjected to high throughput sequencing. To apply this powerful approach for the study of skin biology, we have developed a protocol for the isolation of labeled single cells from neonatal mouse skin. Dispase is used to separate the epidermis from the dermis, with hair follicles remaining in the dermis. Enzymatic digestion then generates single cell suspension, and fluorescently-labeled single cells are sorted into microtiter plates using fluorescence-activated cell sorting (FACS). We have optimized this approach to maximize post-sort cell viability and cDNA purification for scRNA-seq library preparation. Taken together, this approach allows for the collection of high quality scRNA-seq data to analyze the biology of lineage specific cells from neonatal mouse skin.