Extended Data Fig. 7: Transcriptome and CTP analysis in RNAseq of PC9 single cell derived clones.

(a) Distributions of total molecules (UMIs) per clone without drug treatment (DMSO) and with gefitinib drug treatment (+drug). Box plots show medians as central lines. Edges of each box represent the interquartile range (IQR) and whiskers mark either 1.5 × IQR or minima/maxima (if no point exceeded 1.5 * IQR). No removal of outliers was performed in this plot. (b) log2 of total UMIs obtained per clone in both DMSO (x axis) and drug treatment (y axis). n = 195 single-cell derived clonal populations were sampled by at least 10 K UMIs in both treatments. (c) Distribution of clonal CTP values, as measured for n = 195 clones covered in both treatments. (d) gene-gene correlation matrix, showing anti-correlation of two proliferation associated gene modules both in DMSO treated clones (upper triangle) and drug treated clones (lower triangle). (e) Total normalized expression per clone (UMIs per 100 K UMIs) summed from 200 top correlated genes to RPL8 (x axis) and RPL23 (y axis). Color of dots indicates two culturing batches of clones. (f) Expression score per clones for gene modules described in D, computed as log2 ratio of RPL8 / RPL23 total expression (x axis), and is not associated with clonal CTP (y axis). Normalized CTP per clone was computed as log2 ratio of each clone’s CTP to median CTP of the relevant culturing batch. (g) Expression of RPL8 gene module is positively correlated with growth rates. Clones are binned according to the percentage of increment in cells, measured between 5 and 7 days since splitting cells to growth assay (see Methods), and RPL8 module expression is computed as summation of clonal UMIs for 10 genes with highest correlation to RPL8 (units of UMIs per 100 K UMIs). (h) Scatter plots showing the normalized expression of Mucin5B pre- and 24 hours post- 0.5 μM gefitinib treatment. Spearman’s correlation coefficient is indicated for each plot. (i) Cumulative distributions of Spearman’s correlation of individual gene expression to CTP in DMSO treated clones. (Top-left) Showing lowest correlation values obtained between genes and CTP (red), and lowest correlation between genes and a randomized vector of CTP values (orange). (Top-right) Showing highest correlation between genes and CTP (blue), and between genes and a randomized vector of CTP values (lightblue). (Bottom panel) Displaying genes with highest (blue) and lowest (red) correlation to CTP, along with their correlation to randomized CTP vector (lightblue and orange bars). (j) as h, for gefitinib treated clones. Highlighting genes that maintain strong correlation to CTP in both drug treated and untreated clones. (k) Scatter plots showing log normalized expression of selected genes (x axis) and clonal CTP (y axis). (l) Scatter plots showing the normalized expression of ALDH1A3 pre- and 24 hours post- 0.5 μM gefitinib treatment. Spearman’s correlation coefficient is indicated for each plot. Statistical significance for H and L was assessed using a two-tailed Student’s t-test.