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.

Distinct Regulatory Programs Control the Latent Regenerative Potential of Dermal Fibroblasts during Wound Healing

“…To dissect the molecular programs driving divergent fibroblast fates during fibrotic and regenerative healing, we profiled 29,269 single cells from uninjured skin (P28), small wounds at 8 and 14 dpw, and large wounds (divided into central and peripheral domains) at 14 dpw. To increase power for interrogating fibroblast heterogeneity, Hic1:tdT + and tdT À cells were purified by fluorescence-activated cell sorting (FACS) to enrich fibroblasts and wound microenvironment-establishing cells, respectively, and profiled using the droplet-based 10x Genomics system (v.2 chemistry) (Zheng et al, 2017;Stratton et al, 2019b). We selected 12,326 Hic1-tdT + fibroblasts based on Pdgfra, Dpt, and tdTomato expression (Figures S4A-S4C) and performed unsupervised clustering using t-distributed stochastic neighbor embedding (t-SNE) to assess transcriptional heterogeneity within fibroblasts isolated from different wound types (Figure S4D).…”

A subpopulation of embryonic microglia respond to maternal stress and influence nearby neural progenitors

“…By gating for CD11b high and CD45 + (CD45 low-high ; Figure S1B), we collected both CD45 low microglia and CD45 high border-associated macrophages (BAMs), which we reasoned would be helpful for comparison purposes. Enriched microglial cells were processed using the droplet-based 103 Genomics platform (v3 chemistry), as previously described (Stratton et al, 2019), for a combined total of 2,623 sequenced microglia (wild-type and cold stress) from 15 embryos (Figure S1). Despite FACS purification, a proportion (19%) of the sequenced cells were contaminating cells (e.g., NSCs, newly born neurons and glia, endothelial cells, pericytes, etc.)…”

“…Single-cell RNA sequencing library construction and sequencing Single cells were processed using 10x Genomics Chromium Controller and the Chromium Single Cell 3 0 v3 Library & Gel Bead Kit (Product Code: 1000075). Briefly, 8,834 (wild-type) and 9,055 (cold stress) viable PE / FITC double positive single cells were FACS sorted from each treatment and partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs) using 10x GemCode Technology as previously described (Stratton et al, 2019). This process lysed partitioned cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA.…”

Profiling Chromatin Accessibility at Single-Cell Resolution

“…Leading the revolution in single-cell measurement technologies is single-cell RNA sequencing (scRNA-seq), which has imparted significant insights into gene expression. Owing to rapid developments in technology, such as droplet-based microfluidics capable of profiling thousands of single-cells by counting 3′-end of transcripts [4] , [5] , organism-scale atlases have started to detangle transcriptional heterogeneity in cells comprising complex tissues [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] .…”