Fig. 1

Workflow for distribution-based methodology. a Processing of raw images into distributions. Images are segmented based on nuclear staining (blue) and cytoplasmic GFP (green) to yield cytoplasmic fluorescence intensities for each cell (green or grey, if below background). These values are used to estimate a probability distribution for the parameter. b Schematic of single-cell distribution-based methodology. Parameter values are converted into a probability distribution estimate. The distances between each probability distribution are used to assign each condition a point in Euclidean space. Dimensionality reduction is performed using PCA and clustering applied to distinguish effects and categorize the outliers. c NG4 line vector [11]. The BAC-based GFP reporter is driven by the Nanog promoter. d Schematic of siRNA screen as previously described [12]. Pools of siRNA covering the mouse genome are printed onto 55 384-well plates along with controls in triplicate. NG4 cells are reverse transfected and cultured for 3 days under mild differentiation conditions. Cells are fixed, nuclei stained and plates imaged at cell-level resolution