a, b, Ifnb1 mRNA expression and the effect of IFN-β on variation. a, Single-cell expression distributions for the Ifnb1 transcript at each time point (top) after stimulation with PAM (top, green), LPS (middle, black), or PIC (bottom, magenta). Distributions are produced with the density function in R with default parameters, and scaled to have the same maximum density. b, For each of three modules defined in Fig. 1 (core antiviral, top; peaked inflammatory, middle; sustained inflammatory, bottom), shown are bar plots of the fraction of genes (y axis) with a significant change only in α (by a likelihood ratio test, P < 0.01, blue), only in μ (Wilcoxon test, P < 0.01, green), or in both (each test independently, light blue) between the 2 h LPS stimulation and the 2 h IFN-β stimulation separated by whether they increase or decrease during that transition. In each module and condition, the proportion is calculated out of the genes in the module that are significantly bimodal (by a likelihood ratio test) in at least time point during the LPS response and are expressed in at least 10 cells in both conditions. Their number is marked on top of each bar. c, d, Ifnb1 mRNA expression patterns and effect of cycloheximide. c, From top to bottom, population average Ifnb1 mRNA expression (top), single-cell average Ifnb1 mRNA expression (second to top), α (second to bottom) and μ (bottom) estimates for Ifnb1 for each stimulation condition in Fig. 1. Grey star at 6 h for PIC denotes uncertainty due to the small number of cells captured at that time point. d, Shown are box plots of the core antiviral scores (population level, see Supplementary Information) after a 4 h LPS stimulation either where cycloheximide was added at the time of stimulation (right, blue), or during a standard 4 h LPS control (left, green). Core antiviral expression is dramatically decreased by the addition of cycloheximide, suggesting that newly produced protein is needed to initiate the antiviral response. e, Relationship between core antiviral gene expression and Ifnb1 mRNA expression during the LPS, PAM and PIC stimulation time courses and in follow-up experiments. Shown are the expression of core antiviral genes (heat maps: rows, gene; columns, cells) for cells stimulated for 0, 1, 2, 4 or 6 h (left to right) with LPS (top), PAM (middle), or PIC (bottom). Beneath each heat map, grey bars depict the core antiviral score (middle panel, see Supplementary Information) and blue dots show Ifnb1 mRNA expression for each cell in every heat map. f–k, Identifying the precocious cells. f, Core antiviral scores for cells stimulated with LPS, PIC, or PAM. Shown are violin plots of the core antiviral module scores (Supplementary Information, y axis) for each cell from time course experiments (from left: 0, 1, 2, 4 and 6 h) of dendritic cells stimulated with LPS (top), PIC (middle) or PAM (bottom). Two precocious cells (yellow stars, top panel) have unusually high antiviral scores at 1 h LPS (yellow stars, top); similarly precocious cells can be seen in PIC at 1 h and 2 h (orange stars, middle) or in PAM at 2 h (turquoise stars, bottom). g, Precocious cells in all three responses are typical maturing cells. PCA showing the separation between maturing (blue dots) and cluster-disrupted (red dots) cells (top), as well as only maturing (middle) or only cluster-disrupted (bottom) cells (all as also shown in Extended Data Fig. 4d). The precocious cells from each of the responses are marked as stars (colours as in (f)), and all fall well within the maturing cells. h, Precocious cells in all three responses express Lyz1 and do not express SerpinB6b. Shown are mRNA expression distributions for SerpinB6b (cluster disruption cell marker, left) and Lyz1 (normal maturing cell marker, right) in LPS 1 h, PIC 1 h and 2 h, and PAM 1 h (top to bottom). The typical range for expression in cluster-disrupted cells is shaded in red. The precocious cells from each of the responses are marked as stars (colours as in (f)), and all fall well within the maturing cells. i, Normal quantile plots of the expression of genes from the core (cluster I d , left) and secondary (cluster I c , right) antiviral clusters at 1 h LPS. The two precocious cells (yellow stars) express unusually high levels of core antiviral genes (left) but not of secondary genes (right). j, k, The precocious cells are only distinguished by the expression of ∼100 core antiviral genes. Shown are the distributions of scores for each of the first six PCs (right) for samples collected after stimulation with LPS for 1 h with (j) or without (k) the inclusion of core antiviral genes. Precocious cells (vertical red bars), normally distinguished by the third and fourth principle components (j), become indistinguishable from all other cells if the ∼ 100 core antiviral genes are excluded (k) before performing the PCA.