Extended Data Fig. 7: Validation of inferred perturbation integration.

a–c, Perturbation–phenotype association. Spatially resolved visualization of the inferred probabilities indicating the presence or absence of each of the 8 perturbations associated with annotated tumour nodules (Methods). a, A representative sample is displayed. A generalized linear model (GLM) predicts phenotype expression signals based on the estimated probabilities of perturbation presence (Methods). b, Phenotypes are defined as direct target transcripts associated with perturbations such as shKmt2c-Kmt2c and shTrp53-Trp53. Expression data are log1p-transformed. Note that shTrp53 is linked to a GFP peptide barcode and shKmt2c is linked to a RFP barcode (Extended Data Fig. 1). c, Hence we infer shTrp53-GFP-positive phenotype and shKmt2c-RFP-positive phenotype. Representative IHCs for a corresponding ROI on a serial section. Baseline depicts background phenotype marker expression. Data are presented as feature coefficients shown as mean and error-bars depict 3σ confidence intervals. As in Fig. 3c, and d. d, H&E and IHC for GFP, RFP, GS. Three representative FFPE samples from a single RUBIX experiment with two animals were sectioned and stained for H&E (see Extended Data Fig. 2). GFP and RFP IHC staining was performed on two individual serial sections. GS IHC staining was performed on serial sections. GFP and RFP were embedded in perturbation plasmids as orthogonal barcodes (see Methods and Extended Data Fig. 1). GS is a well-known marker for liver WNT/mtCtnnb1-signalling activity (see Fig. 3d). e, GFP and RFP detection for the same hepatocellular tumour nodules to spatially map peptide barcode combinations associated with introduced perturbations. Left: H&E. Zoom-in: GFP and RFP respectively. Note that one tumour nodule is positive for RFP alone whereas the other tumour nodule is positive for both GFP as well as RFP peptide barcodes. A representative example is shown. Data is derived from 324 nodules across 6 topographically separated regions used for 10X Visium from a single RUBIX experiment with two animals. Mapping GFP and RFP IHC data is derived from 3 corresponding sections from a single RUBIX experiment with two animals.