Journal: Nature Communications

Article Title: Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels

doi: 10.1038/s41467-018-06891-x

Figure Lengend Snippet: VSMCs show heterogeneous expression of genes implicated in cardiovascular disease. a Scatter plots showing the mean-variance relationship of log 2 -transformed normalised expression levels for each gene, with colour-highlighting of genes showing highly variable expression in the aortic arch (AA, red, top panel) and descending thoracic aorta (DT, yellow, lower panel). b Bar graph showing the implication of the identified highly variable genes (HVGs) in AA (red), DT (yellow) or both regions (green) in functions related to VSMC biology based on published literature (see Methods for details). c Dot plot showing log 2 -transformed normalised counts detected in individual VSMCs from AA (red) and DT (yellow) for selected genes that show variable expression across single cells. d , e t-SNE plot visualising a 10X Chromium dataset generated from 2846 unselected cells (gated as live using Zombie NIR staining and singlets using doublet discrimination) from the whole aortas of three tamoxifen-labelled Myh11-CreERt2/Confetti animals (pooled). d Clusters generated using graph-based clustering are colour-coded as indicated and adventitial (Adv), endothelial (EC) and VSMC (VSMC) populations are labelled. e Log-transformed expression levels of selected HVGs identified in AA and DT populations based on Fluidigm C1 data ( Rgs5 , Irf1 , Atf3 , Nfkbia ), shown using a scale from light to dark grey

Article Snippet: Raw sequencing reads were processed and aligned to the GRCm38 mouse genome through the 10X Genomics cellranger pipeline (v2.0 for VSMC-derived plaque cells and v2.1 for whole aorta cells and VSMC-lineage cells from healthy vessels).

Techniques: Expressing, Transformation Assay, Generated, Staining

Journal: Nature Communications

Article Title: Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels

doi: 10.1038/s41467-018-06891-x

Figure Lengend Snippet: A subset of medial cells express Sca1. a t-SNE plot visualising the 10X Chromium dataset of unselected cells from whole aortas (shown in Fig. ), with log-transformed expression levels of Ly6a/Sca1 in each cell colour-coded on a scale from light to dark grey. b Representative confocal images of GFP-positive cells isolated from the media (top row) or adventitia (Adv, middle row) of Sca1-GFP animals after immunostaining for aSMA. Signals for GFP (green), anti-aSMA (magenta) and nuclear DAPI (white) are shown individually and merged as indicated. The lower row shows GFP-negative medial cells isolated from wild-type animals and stained with isotype IgG as a control for staining and GFP detection. Scale bars are 10 µm. c Dot plot showing the percentage of aSMA– and aSMA+ cells in sorted GFP+ adventitial (Adv, red dot, n = 1) and medial cells (green dots, n = 4) from Sca1-GFP animals. Data for individual replicates and their mean values are indicated and error bars show s.e.m.

Article Snippet: Raw sequencing reads were processed and aligned to the GRCm38 mouse genome through the 10X Genomics cellranger pipeline (v2.0 for VSMC-derived plaque cells and v2.1 for whole aorta cells and VSMC-lineage cells from healthy vessels).

Techniques: Transformation Assay, Expressing, Isolation, Immunostaining, Staining, Control

Journal: Nature Communications

Article Title: Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels

doi: 10.1038/s41467-018-06891-x

Figure Lengend Snippet: The transcriptional signature of S+L+ cells from healthy vessels is expressed in VSMC-derived plaque cells. a t-SNE visualisation of 10X Chromium dataset (3314 cells) of lineage-positive cells isolated from aortic plaques and the underlying media in Myh11-CreERt2/Confetti mice that were first tamoxifen-treated and then fed a cholesterol-rich diet for 14 or 18 weeks (tissue from three and two animals respectively was pooled for the analysis). The cells are grouped into a large population composed of 9 clusters, including one enriched for chondrocytic genes (cluster 8, magenta), and a smaller, distinct population, which was enriched for macrophage genes (cluster 9, pink). b t-SNE plot of 10X Chromium dataset from a with Ly6a/Sca1 expression colour-coded from light to dark grey. c , d t-SNE plot of 10X Chromium from a , with expression scores for cVSMCpos ( c ) or cVSMCneg ( d ) genes colour-coded on a blue to red gradient. e Violin plots showing the expression score of cVSMCpos genes in cells mapping to each cluster shown in a . f Confocal image of a cryosection of the descending thoracic aorta from a Myh11-CreERt2/Confetti/ApoE -/- mouse fed a cholesterol-rich diet for 30 weeks and immunostained for Sca1. Signals for fluorescent Confetti proteins (GFP, green; RFP, red; YFP, yellow and CFP, blue) are shown in i–iii, nuclear DAPI (white) is shown in all panels, and anti-Sca1 (magenta) is shown in i, ii, and iv. The region outlined in i is magnified in ii–iv, with arrows pointing to cells that are double-positive for the Confetti lineage label (RFP) and Sca1. Image in i is a maximum projection of 16 z-slices (2 µm each) and ii–iv show a single 2 µm Z-slice. Scale bars are 40 μm (i) or 15 μm (ii–iv)

Article Snippet: Raw sequencing reads were processed and aligned to the GRCm38 mouse genome through the 10X Genomics cellranger pipeline (v2.0 for VSMC-derived plaque cells and v2.1 for whole aorta cells and VSMC-lineage cells from healthy vessels).

Techniques: Derivative Assay, Isolation, Expressing