Extended Data Fig. 4: OverCITE-seq identifies ORFs and their transcriptional effects.
From: A genome-scale screen for synthetic drivers of T cell proliferation
a, Quality parameters of cells as identified by gel bead barcodes. Negative, singlets and doublets are assigned based on cell hashing. b, Proportion of stimulated and resting T cells among cells assigned to each ORF. Chi-squared test p-values are shown for ORFs with significantly shifted (uneven) distributions of stimulated and rested cells. c, Cell-cycle corrected scaled expression of the overexpressed gene in the cells transduced with the respective ORF and negative control (tNGFR). Two-sided Wilcoxon test p-values shown above the violin plots indicate the statistical significance of gene expression level between specific ORF and tNGFR-transduced T cells. Box shows 25–75 percentile with a line at the median; whiskers extend to maximum and minimum values. N = 71 (ADA), 147 (AHCY), 190 (AHNAK), 119 (AKR1C4), 124 (ATF6B), 179 (BATF), 137 (CALML3), 189 (CDK1), 129 (CDK2), 236 (CLIC1), 84 (CRLF2), 91 (CXCL12), 88 (CYP27A1), 129 (DBI), 26 (DCLRE1B), 261 (DUPD1), 25 (FOSB), 119 (GPD1), 124 (GPN3), 199 (IFNL2), 60 (IL12B), 70 (IL1RN), 156 (ITM2A), 74 (LTBR), 88 (MRPL18), 167 (MRPL51), 107 (MS4A3), 69 (NFYB), 355 (NGFR), 261 (RAN), 182 (SLC10A7), and 56 (ZNF830) single cells. d, Expression of all ORF genes by cells assigned each ORF. Each row is z-score normalized. e, Distribution of individual ORF frequencies in clusters. Numbers of ORF cells and the chi-squared test residuals are displayed. Chi-squared test p-values indicating whether ORF distribution in each cluster significantly differs from overall ORF distribution are shown on top of the plot. Proportions of stimulated and resting T cells in each cluster are shown underneath the cluster label. f, g, Spearman correlations between transcriptional profiles of selected ORF cells in resting (f) and stimulated (g) populations. h, Fold change of top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORFs with the strongest transcriptional changes (compared to tNGFR cells) are shown. i, Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p < 0.05). For all genes, we display log2 fold-change of each ORF (stimulated) to tNGFR (resting), normalized to log2 fold-change of tNGFR (stimulated) to tNGFR (resting). Genes of interest in each cluster are labelled. j, Mean TCR clonotype diversity in ORF cells.
