Figure 1.

Overview of experimental design and library construction

(A) Schematic of lentiviral overexpression vector.

(B) Composition of the cancer driver ORF library, encompassing major signaling pathways, oncogenes and stemness regulators involved in oncogenesis and cancer progression.

(C) Schematic of experimental framework for evaluation of effects of cancer driver overexpression in developing teratomas and reinjected tumors: Individual cancer driver ORFs are cloned into the barcoded ORF overexpression vector, packaged into lentivirus then pooled for transduction of hESCs. Transduced cells are then injected subcutaneously into Rag2^−/−;γc^−/− immunodeficient mice to form the round 1 teratomas. Teratoma growth is assayed by caliper-based measurement of approximate elliptical area of the tumor. Once the teratoma size reaches a threshold value, teratomas are excised and assayed by single-cell RNA-seq and histology, and a fraction of cells are reinjected in Rag2^−/−;γc^−/− immunodeficient mice to form round 2 tumors for further enrichment of drivers. Round 2 tumor growth is also monitored via caliper-based measurements and at the endpoint tumors are assayed via scRNA-seq and histology.