High-throughput chemical screens typically employ coarse assays, e.g. cell survival, limiting what can be learned about mechanisms of action, off-target effects, and heterogeneous responses. Here we introduce sci-Plex, which uses ‘nuclear hashing’ to quantify global transcriptional responses to thousands of independent perturbations at single-cell resolution. As a proof-of-concept, we applied sci-Plex to screen 3 cancer cell lines exposed to 188 compounds. In total, we profiled ~650,000 single-cell transcriptomes across ~5,000 independent samples in one experiment. Our results reveal substantial intercellular heterogeneity in response to specific compounds, commonalities in response to families of compounds, and insight into differential properties within families. In particular, our results with HDAC inhibitors support the view that chromatin acts as an important reservoir of acetate in cancer cells.
* co-first authors ** corresponding authors