Fig. 5: Live-seq links transcriptomic state with subsequent functional analysis on the same cells.

a, Schematic illustrating the coupling of Live-seq with live-cell imaging. RAW cells were first subjected to Live-seq and subsequently exposed to LPS while tracking Tnf-mCherry fluorescence by time-lapse imaging. b, A linear regression model was used to predict the slope (the rate of Tnf-mCherry fluorescence intensity increase) calculated from data shown in Extended Data Fig. 9b and Supplementary Fig. 7 on the basis of ground-state gene expression data recorded by Live-seq. The most variable genes from both Live-seq and scRNA-seq data were used, whereas genes with dispersion <0.1 in Live-seq samples were removed (Methods). c, Basal Nfkbia expression, as determined by Live-seq, anticorrelates with the rate of Tnf-mCherry fluorescence intensity increase. R² and FDR are listed. d, The expression of Nfkbia and Tnf is highly correlated in conventional scRNA-seq data. P = 2 × 10⁻⁵², two-sided F-test. e, Validation of Nfkbia expression as a predictor of the extent by which a macrophage will respond to LPS using a Nfkbia-BFP reporter (Methods). n = 91, R² = 0.11, P = 0.0008, two-sided F-test; Pearson’s r = −0.34, P = 0.0008. Another biological replicate is shown in Extended Data Fig. 9h. a.u., arbitrary units. f, The (basal) cell cycle S phase score of Live-seq samples (inferred from respective transcriptomes) anticorrelates with the rate of Tnf-mCherry fluorescence intensity increase, suggesting that cells in S phase respond weaker to LPS treatment. R² and FDR values are listed. The error band (c–f) represents the s.d. g, Validation of cells in S phase responding weaker to LPS exposure using a Fucci cell cycle indicator (Extended Data Fig. 10b,c and Methods). n = 32 cells over two independent experiments. Error bars represent the mean ± s.d. and P values were determined by a two-sided Wilcoxon rank-sum test.