a) Hierarchical clustering of single-cell transcriptomes with unambiguously assigned gRNA (n = 5,798) based on all genes included in the TCR activation signature. Clustering for cells and genes used the Pearson correlation, and the z-score of expression is displayed along with the stimulation state for each cell (left column). b) Hierarchical clustering of median gene expression values aggregated across cells expressing gRNAs for the same target gene (n = 107). c) Analytical procedure for assigning each cell to a specific position on a spectrum defined by the CROP-seq transcriptomes of non-targeted cells in the naive and the anti-CD3/CD28 stimulated cell state. In a first step (left), a matrix of synthetic transcriptome signatures (Z) is built by linear combination of the transcriptomes in the two defining cell states (μ_A, μ_B). In a second step (right), the position of the Z matrix that shows the maximum Pearson correlation with the transcriptome of the cells (E matrix) is taken as the cell's position along the spectrum of cell states. d) Correlation (row-wise z-score) of single-cell transcriptomes with a matrix comprising synthetic mixtures of transcriptome profiles between the median of non-targeted cells in both conditions (values close to one reflect similarity with anti-CD3/CD28 stimulated cells). Additional data on the transcrip-tome quality for each cell (unique reads per cell) and the overall correlation performance are shown as columns and reveal no relation-ship with the inferred position of the corresponding cells. All cells were ordered by the inferred signature value (position with maxi-mum correlation), rather than being clustered as in panel a. e) Same as in panel c, but for cells grouped by gRNA target genes.