Journal: Nature Communications

Article Title: Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling

doi: 10.1038/s41467-017-01560-x

Figure Lengend Snippet: Acquisition of molecular heterogeneity in the mammary epithelium during puberty. a Schematic diagram of the mammary gland at pre-puberty (2 weeks; red), puberty (5 weeks; green, with TEBs in dark green) and adulthood (10 weeks; black). b Multidimensional scaling plot of scRNA-seq profiles. Total epithelial cells were collected at pre-puberty, puberty, and adulthood and profiled using the Fluidigm C1 platform. Distances on the plot correspond to leading log2-fold changes between cells. The first plot dimension ( x -axis) clearly separates pre-pubertal cells from those in puberty or adult glands. Pre-puberty, 12 mice; puberty, 6 mice; adult, 4 mice per sort; three independent experiments. c Gene expression heatmap of the same 460 single cells shown in b . Rows correspond to the top 500 DE genes across the three developmental stages. Columns correspond to cells colored by stage as in a and b , indicated by bars at the top. Major gene clusters I to VI are highlighted in the side column; Supplementary Table lists genes in these clusters. Selected genes are shown for clusters I, II, and IV, comprising genes specifically expressed at 2 weeks. Heatmap values are log expression scaled by row (red = high expression; blue = low expression). d Ternary plot showing relative expression of selected genes in the three developmental stages. For each gene, expression was averaged over all cells at each stage. Most genes known to be luminal-specific (purple) are strongly upregulated in cells from 5 and 10-week-old glands while different basal-specific genes (sky blue) are expressed at different developmental stages. Genes correspond to the top 1000 DE genes from Fu et al. 2015 . e Violin plots showing gene expression of selected genes for each cell by developmental stage. Vertical axis shows expression as log2-RPKM

Article Snippet: Read pairs from the Fluidigm C1 scRNA-sequencing were mapped to the mm10 mouse genome using the Subread aligner and assigned to genes using featureCounts and Rsubread’s built-in refSeq annotation.

Techniques: Expressing

Journal: Nature Communications

Article Title: Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling

doi: 10.1038/s41467-017-01560-x

Figure Lengend Snippet: Identification of multiple subsets in pubertal and adult mammary glands. a Heatmap showing hierarchical clustering of 343 epithelial cells from pubertal (green) and adult (black) mammary glands based on their expression of lineage-specific genes (200 basal and 200 luminal genes; see Supplementary Table ). Columns correspond to cells colored by stage, indicated by bars at the top. The cells shown are the same 5 and 10-week-old cells as in Fig. , c and Supplementary Fig. . Seven major cell clusters are indicated (gene lists shown in Supplementary Table ). Basal genes = blue; luminal genes = purple; red = high expression; blue = low expression. b Multidimensional scaling plot of scRNA-seq profiles. These are the same as in Fig. but cells expressing Cd55 (RPKM > 40) are highlighted as blue triangles. Cd55 + cells are present at all three stages but pre-pubertal glands harbor only rare cells (circled in the plot). c CD55 expression within epithelial subsets isolated from mammary glands in pre-puberty (2 wks) (left), puberty (5 weeks) (center), and adult (10 weeks) (right). Representative dot plots of CD29 vs. CD24 expression in Lin – cells (see gating strategy in Supplementary Fig. ) are shown on the left, while CD29 vs. CD55 expression in either the luminal (top) or basal (bottom) compartment is shown on the right ( n = 3 independent experiments per time-point: 12 mice per experiment for pre-puberty, 6 mice per experiment for puberty, and 4 mice per experiment for adult). d The CD14/CD55 flow cytometric plots of freshly sorted Lin – CD29 lo CD24 + (luminal) mammary epithelial cells isolated from 10-week-old FVB/N mice (representative of 5 independent experiments, 4 mice per experiment). e Hierarchical clustering of 76 purified luminal cells based on Cd55 and Cd14 gene expression (Fluidigm C1 platform; see Supplementary Fig. ). Red = high expression; blue = low expression. f Clonogenic activity of CD14 – CD55 – , CD14 – CD55 + , CD14 + CD55 – , and CD14 + CD55 + luminal (CD29 lo CD24 + ) subsets from 10-week-old FVB/N mice, plated in Matrigel (1000 cells per population). Representative of three independent experiments, 4 mice per experiment. P -value (unpaired t -test) <0.0001. Scale bar; 1000 µm

Article Snippet: Read pairs from the Fluidigm C1 scRNA-sequencing were mapped to the mm10 mouse genome using the Subread aligner and assigned to genes using featureCounts and Rsubread’s built-in refSeq annotation.

Techniques: Expressing, Isolation, Purification, Activity Assay