LPS and IFNγ both generate stimulus-specific de novo enhancers in human macrophages; however, only IFNγ-induced enhancers are durable. (A) Schematic of experimental design: Human macrophages were stimulated with either IFNγ (100 ng/ml), LPS (100 ng/ml), or LPS in the presence of 1 µM ruxolitinib for 8 h. Cells were subsequently washed and cultured for an additional 88 h. H3K4me1 CUT&Tag was performed at each time point. (B) Heatmap of Z-scored reads within H3K4me1 peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Top enriched motifs in clusters from B. (D) Box/whisker plot quantifying log2 cpm of reads within IFNγ-induced peaks before and after cytokine washout. (E) Box/whisker quantifying log2 cpm of reads within LPS-induced peaks before and after cytokine washout. (F) Box/whisker quantifying log2 cpm of reads within peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (G) Z-scored heatmap of reads within CUT&Tag peaks of only IFNγ-induced peaks before and after cytokine washout. (H) Boxplot of log2 cpm of reads within peaks for each cluster identified in G. (I) Examples of genome browser tracks for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. *P < 0.05; ****P < 0.0001.