(A) Histogram of the DNA content of RPE-fluorescent, ubiquitination-based cell cycle indicator (FUCCI) cells. RPE-FUCCI cells were stained with Hoechst 33,342 for 30 min before fluorescence activated cell sorting (FACS) analysis. Four different gates were set based on FUCCI fluorescence, gating G1, early S, S, and G2/M phase cells. For each FUCCI-based gate we calculated the average Hoechst fluorescence intensity, relative to the intensity of G1 phase cells. Shown is one representative of three experiments. p-Value is based on a one-tailed unpaired Student’s t-test. p-Value is indicated as *** (p < 0.001). (B) Fluorescence microscopy time traces of RPE-1 cells expressing FUCCI-G1 and FUCCI-G2 markers. Asynchronously growing RPE-FUCCI cells were imaged every 5 min and the average nuclear intensities for both FUCCI markers were measured (red and green lines, respectively). Dark red and green lines represent the average of 30 cells, light red and green lines represent individual cells of one representative experiment of three experiments. For quantification of FUCCI-G1 fluorescence during G1 phase, cells that entered S phase within the 10 hr window post-mitosis were excluded, because the FUCCI-G1 reporter is rapidly degraded upon S phase entry. S phase entry is identified based on an increase in FUCCI-G2 fluorescence (Figure 1A–C and Figure 1—figure supplement 1A). See Supplementary file 2 for the number of cells included in each condition. (C) Measurement error in the G1 phase cell cycle time due to cell-to-cell variability in FUCCI-G1 fluorescence intensities. We calculated the standard deviation of the G1 phase cell cycle time (see Materials and methods). Since cell-to-cell variability in FUCCI-G1 fluorescence increases at later time points in G1 phase (Figure 1D and (B)), the measurement error of G1 phase cell cycle time increases at later time points in G1 phase. Red squares are individual data points. (D) Microscopy-based analysis of FUCCI fluorescence intensities in RPE-FUCCI cells. Early S phase cells were identified based on the presence of high FUCCI-G1 and low FUCCI-G2 fluorescence (yellow dots), and the FUCCI-G1 fluorescence intensity of early S phase cells was recorded (see Materials and methods). A representative experiment is shown (three experiments were performed with at least 700 cells quantified per experiment). (E) FACS plots of FUCCI fluorescence after different durations of Taxol treatment. To quantify the increase in FUCCI-G1 fluorescence over time, asynchronously growing RPE-FUCCI cells were treated with Taxol for the indicated durations to arrest cells in mitosis, thus preventing new cells from entering G1 phase. Over time, FUCCI-G1 fluorescence increases which results in the gradual loss of cells with low FUCCI-G1 fluorescence. The lowest FUCCI-G1 fluorescence intensity after a 1, 2, or 4 hr incubation with Taxol were identified (dotted lines), and used to calculate the FUCCI-G1 fluorescence at various times during G1 phase relative to early S phase (see Materials and methods). (F) Comparison of the relative FUCCI-G1 fluorescence intensities as determined by the polynomial equation (Figure 1D) and by Taxol treatment of cells followed by FACS (see panel S1E) at 1, 2, and 4 hr after mitosis. Bars and error bars indicate average ± SEM of three experiments. (G–I) Comparison of normalized read counts in G2/M from three sequencing experiments. Each dot represents the expression of one gene averaged over all cells in G2/M phase. Dotted red line indicates identical read counts in two experiments. (J) Histogram showing the position in the cell cycle of all cells subjected to single-cell RNA sequencing (scRNA-seq). The position in the cell cycle was determined based on the FUCCI-G1 fluorescence intensity as measured by FACS. (K) Gene Ontology term analysis of genes downregulated in G1 phase compared to G2/M phase. For this analysis, cells were grouped into either G2/M or G1 phase based on FUCCI fluorescence. (L) Pie chart showing the fraction of all genes with known cell cycle functions (derived from Cyclebase 3.0) that are up- or downregulated in G1 phase compared to G2/M phase, or that show no change in expression. (M) Single-cell trajectory of the mitosis-to-G1 (M-G1) phase transition constructed by Monocle2. Colors indicate the cell cycle position based on FUCCI-G1 fluorescence. (N) Differential transcriptome analysis of G1 phase versus G2/M phase RPE-FUCCI cells, aligned based on Monocle2 trajectory inference.