Extended Data Fig. 2: Identification of cell-cycle independent transcription variation of HCT116, H1299 and WI38 single cells.

a-c, Normalized pooled expression in clonal populations (x-axis) and single-cells (y-axis) in WI38 (left), H1299 (center) and HCT116 (right) cells. Expression values computed as log₂ of UMIs / 10 K UMIs. Genes with high differential expression in each system are displayed by red dots and annotated. n (WI38, HCT116) = 27,052, n (H1299) = 17,698. d, Distributions of total number of molecules per cell in inferred cell-cycle metacells of HCT116. Colors are as in Fig. 1b. e, log₂ total expression signatures (left) and ratio of cell-cycle phases (right) in WI38 cells. Right panel shows only cells that were annotated as replicating in left panel. f, as in e for H1299 cells. g, as in e for HCT116 cells. A full list of genes used in this assay for all cell lines is in Supplementary Table 1. h, Illustration of expression randomization in each cell according to cell-cycle based cell-cell similarity graph. i, Showing scRNA gene profiles correlation with EpCAM expression, controlled by each gene total expression (left), with a running median shown in red. Following subtraction of the trendline, correlations are generally independent of gene sampling depth (right). j, Matrix of gene-gene correlations in HCT116 cells before (upper triangle) and after (lower triangle) cell-cycle based randomization. Showing selected cell-cycle related (Supplementary Table 1) and unrelated (Supplementary Table 3) gene modules. Number of analyzed cells defined in Extended Data Fig. 1c. k, Maximal correlation value of each gene with another gene before (x-axis) and after (y-axis) cell-cycle based randomization. Loss of correlation (blue dots) indicates that the co-expression patterns of this gene were independent of the cell-cycle, thus eliminated by the randomization. l-m, as in j-k, for NCI-H1299 cells. n-o, as in j-k, for WI38 cells.