Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

Abstract

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

Immune modulation of hair follicle regeneration

“…Single-cell RNA-Seq library construction All procedures received prior approval from the University of Calgary Health Sciences Animal Care Committee and were completed in accordance with the Canadian Council of Animal Care guidelines (Protocol AC-140019). Single cells were isolated and pooled from N = 3 female Hic1 CreERT2 :Rosa tdTomato (C57BL/6J background, treated with tamoxifen at postnatal days 3 and 4) mice at postnatal day 28 as previously described 158 . Briefly, anagen backskin was dissected and enzymatically dissociated with dispase for 20 min at 37 °C to remove the epidermis.…”