Extended Data Fig. 5: Validation of the FUCCI cell model.

a, Images from a time-lapse microscopy series of FUCCI cells show that the length of the cell cycle phases marked by the different colours of the FUCCI tags are 10.8 h for G1, 2.6 h for S-transition, and 11.9 h for G2. U2OS FUCCI cells allow monitoring of the cell cycle by expressing two fluorescently tagged cell cycle markers: CDT1 during G1 phase (red), GMNN during S and G2 phases (green), and both during the S-transition (yellow). Scale bars, 10 μm. b, The polar coordinate model transfers the FUCCI marker information into a linear model of pseudotime. The tagged CDT1 (red) and GMNN (green) log-intensities are displayed across pseudotime, which they are used to calculate. The solid line is the moving average across 100 cells (264,863 total), and light shading fills between the 25th and 75th percentiles of intensity. c, Cell division time can also be predicted from scRNA-seq data, and so we performed a comparison between the pseudotime calculated using FUCCI marker intensities in this work (x-axis, colour mapping) and the pseudotime inferred from scRNA-seq markers using the tool Peco⁵⁹ (y-axis). The most notable feature of this comparison is the more continuous nature of the pseudotime calculated using FUCCI marker intensities, indicating that the experimental FUCCI marker intensities provide a better basis than inferred pseudotime for the integration of single-cell proteomic and transcriptomic data. d, CNVs identified in single-cell transcriptomic data correlate to the cell cycle, thus validating the FUCCI model. e, BrdU incorporation (blue) into FUCCI cells. Incorporation of the synthetic nucleotide BrdU occurs during DNA replication in S-phase of proliferating cells and remains through the rest of the cell cycle. Here, G1 cells with red nuclei show no incorporation of BrdU, whereas most of the S and G2 cells with green nuclei show incorporation of BrdU (bottom left and centre; G1, n = 45; S-trans, n = 32; S and G2, n = 73 independent cells). For box plots: centre line, median; box, Q1 and Q3; whiskers, 1.5× IQR below Q1 and above Q3; points, outliers. Based on this, we can estimate the proportion of cells in each phase (bottom right). These results show the validity of the FUCCI model. Scale bar, 10 μm.