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. 2022 Jul 18;11:e80167. doi: 10.7554/eLife.80167

Figure 2. scRNA-seq reveals ASC heterogeneity and maps adipogenic trajectories in mouse iBAT.

(A) t-SNE plot of 28,691 lineage marker negative (Lin-) cells from iBAT of control mice and mice exposed to cold for four days. Clustering identified eight major clusters, highlighted in different colors. ASC, adipose tissue stromal cell; VEC, vascular endothelial cell; VSMC, vascular smooth muscle cells; Prolif/Diff, proliferating/differentiating cells. DEGs that define these clusters are in Supplementary file 1. (B) t-SNE plot from (A) split into cells from the separate treatments (CONTROL and COLD). Circles highlight cold-induced clusters. (C) t-SNE plot of 19,659 re-clustered ASC and Prolif/Diff cells from (A). The t-SNE plot and clustering identified six clusters. Prolif/Non-diff, proliferating/non-differentiating; Prolif/Diff, proliferating/differentiating. DEGs that define these clusters are in Supplementary file 2. (D) Violin plots of log2 expression levels of select marker genes for individual clusters from the CONTROL and COLD data presented in (C). (E) t-SNE plots displaying the log2 expression levels for genes involved in adipogenic differentiation from the CONTROL and COLD data presented in (C).

Figure 2.

Figure 2—figure supplement 1. Schematic diagram of single-cell library generation and processing.

Figure 2—figure supplement 1.

(A) Schematic of one single-cell experiment from cohorts of control and cold-exposed mice. Mouse iBAT was harvested and digested into the stomal vascular fraction (SVF). These cells were separated into lineage marker positive (Lin+) and negative (Lin-) cell fractions with magnetic bead cell separation (MACS). Single-cell libraries were prepared from these four cell fractions. (B) Method of demultiplexing and data analysis for the scRNA-seq libraries. Analysis for C57 and Adrb1 KO libraries differed slightly due to method of library generation. (C) Summary of the single-cell libraries presented in this paper. Note that each row of the table corresponds to two scRNA-seq libraries: Lin+ and Lin-.
Figure 2—figure supplement 2. scRNA-seq analysis of Lin- cells from iBAT of control and cold-exposed mice.

Figure 2—figure supplement 2.

Related to Figure 2. (A) t-SNE plot of Lin- data displaying the log2 expression levels for genes commonly used to isolate adipocyte precursors and markers for proliferating/differentiating cells. (B) t-SNE plot of Lin- data with clustering at different resolutions, resulting in different numbers of clusters. (C) Volcano plots of DEGs between cells from control and cold-exposed mice for the three ASC subtypes. Numbers displayed on the plot are the number of genes with an adjusted p-value less than 0.05 and the absolute value of the fold change greater than 0.5.