biostudies-literatureDini AAcademy of FinlandSyöpäsäätiöAcademy of Finland (Suomen Akatemia)Sigrid Juséliuksen Säätiö432-442https://www.ebi.ac.uk/biostudies/studies/S-EPMC11867973biostudies-literatureUnknown21(3)The gene-regulatory dynamics governing drug responses in cancer are yet to be fully understood. Here, we report a pipeline capable of producing high-throughput pharmacotranscriptomic profiling through live-cell barcoding using antibody-oligonucleotide conjugates. This pipeline combines drug screening with 96-plex single-cell RNA sequencing. We show the potential of this approach by exploring the heterogeneous transcriptional landscape of primary high-grade serous ovarian cancer (HGSOC) cells after treatment with 45 drugs, with 13 distinct classes of mechanisms of action. A subset of phosphatidylinositol 3-OH kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) inhibitors induced the activation of receptor tyrosine kinases, such as the epithelial growth factor receNature chemical biologyA multiplex single-cell RNA-Seq pharmacotranscriptomics pipeline for drug discovery.PMC11867973349787333583288475336449271845Piki ESharma SRaivola JDini AMurumagi ABarker HUngureanu DfalseA multiplex single-cell RNA-Seq pharmacotranscriptomics pipeline for drug discovery.The gene-regulatory dynamics governing drug responses in cancer are yet to be fully understood. Here, we report a pipeline capable of producing high-throughput pharmacotranscriptomic profiling through live-cell barcoding using antibody-oligonucleotide conjugates. This pipeline combines drug screening with 96-plex single-cell RNA sequencing. We show the potential of this approach by exploring the heterogeneous transcriptional landscape of primary high-grade serous ovarian cancer (HGSOC) cells after treatment with 45 drugs, with 13 distinct classes of mechanisms of action. A subset of phosphatidylinositol 3-OH kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) inhibitors induced the activation of receptor tyrosine kinases, such as the epithelial growth factor rece2025-01-01T00:00:00Z2025 Mar2025-04-03T23:36:29.89Z2025-04-03T23:36:29.89ZS-EPMC118679733948247010.1038/s41589-024-01761-8