Figure 2.

Pseudo-Timing Reveals Dynamic Signaling Pathway Activity over the Course of Epidermal Differentiation

(A) Combining scID-seq with FACS-based sorting on ITGB1 levels. Cells were immuno-stained with fluorescent ITGB1 antibodies in combination with a panel of 70 Ab-DNA conjugates, FACS sorted based on their ITGB1 levels and subjected to scID-seq.

(B) scID-seq distinguishes ITGB1+ and ITGB1low sorted cells based on known epidermal basal, differentiation, and cell-cycle markers. Distributions of normalized and scaled scID-seq counts of selected proteins verified the separation of the ITGB1+ and ITGB1low populations.

(C) Principal-component analysis on known markers orders epidermal cells on their renewal and differentiation status. Top panel: markers used for the temporal ordering, color intensity represents scaled antibody counts. Bottom panel: cells were ranked on their (scaled) PC1 loading. Vertical lines indicate the 10-cell bins used to smoothen the data in subsequent analyses.

(D) Dynamics of the markers used for PCA, ordered by pseudo-time (scaled PC1 loading) after smoothening. Data points indicate 10-cell bin mean, and solid lines represent model fit of the data (third-order polynomial regression).

(E) Dynamics of two independent phosphorylated Akt/PKB antibodies over pseudo-time.

(F–H) Dynamics of antibodies reflecting the JAK-STAT, WNT, and BMP signaling pathways over pseudo-time.