Fig. 5

Verification of consensus temporal gene signature. A Experimental design for the Pan T-cell Verification Set. We performed RNA Sequencing of Pan T-cells from 4 healthy donors at 5 different time points after anti-CD3/anti-CD28 activation (6 to 72 h) and of unactivated (0 h) T-cells. In addition, we sequenced Pan T-cells for the same time points without activation as negative controls. B Depicted are the fractions of Pan T-cell populations before activation. 200,000 cells were analyzed using seven human blood donors (see Additional file 1 for details). C For each contrast (6 to 72 h of activation and without activation vs. 0 h), we performed a DGEA. The brown and blue bar plots depict the number of DE genes (FDR < 0.05) for each contrast that is up- (brown) or down-regulated (blue) under activated (act), unactivated (neg. ctrl) conditions, or both (act and neg. ctrl). Gray bar plots show the number of DE genes under activated and unactivated conditions without consistent log2 fold change. D Hierarchical clustering of DE genes from the Pan T-cell Verification Set in activated and unactivated conditions. Only genes from the consensus signatures that passed the activation kinetics of both Verification Sets are shown. Euclidean distance with Ward clustering was applied to visualize the similarity between samples. Each column represents a sample, each row represents a gene. The y-axis depicts the standardized median CPM expression values of genes from samples with identical analysis time points. Bottom panel: For each contrast and condition, boxplots of log2 fold changes are shown. E For the Pan T-cell Verification Set the temporal expression pattern of genes from the consensus signatures that passed the 2 Verification Sets (activation and negative control kinetics) are shown. CPM values were z-score standardized. Identity metagenes are highlighted with colored horizontal bars (M1, M3, and M5). The red-colored time points indicate the maximum centroid threshold (see the “Methods” section). Only metagenes with more than 10 genes are shown. F Flowchart depicting the number of genes passing each filter step