Supplementary Figure 3: Validation of DSP with oligo-conjugated antibodies based bulk RNA expression and correlation across replicate ROIs.

a, Additional data from experiment presented in Fig. 1c. 23 core FFPE cell pellet array stained with CD3, CD45, and PanCK fluorescently-conjugated antibodies and SYTO13 nuclear dye, along with a mix of 41 oligo-conjugated antibodies. N=2 replicate ROIs across n=3 ROI areas (50 µm, 100 µm, and 300 µm diameter circles) were profiled (n=6 total technical replicates). Y-axes are scaled according the expression range for each antibody and each ROI size. nCounter counts from 100 ng purified RNA (see methods) were used to estimate which cell lines are positive (> 100 counts) or negative (<10 counts). Targets with nCounter counts between 10 and 100, targets expected to be expressed in all cell lines (housekeeper), or antibodies with no specificity (IgG controls) were labeled as “Unknown” expression. Box plots indicate the median, upper and lower quartiles with whiskers extending to the minimum and maximum values. Box plot were used for these n=2 studies to clearly show the replicate datapoints. b, Unsupervised clustering of scaled data across all ROIs. Data was scaled to characterize general expression patterns for each ROI, while considering variable ROI sizes. Average R² correlations across the 6 ROIs for each cell line were determined by comparing the first replicate of the 100 µm ROI against the 5 other ROIs. These 5 independent R² values were average to obtain the values shown across the heatmap annotation row [Avg R²]. These average R² values were all greater than 0.96, with a range from 0.963 (A549) to 0.999 (MOLT-4). Expected clustering of similar cell line types was also seen with clustering of the leukemia and lymphoma cell lines (Jurkat, CCRF-CEM, HuT 78, MOLT-4, K-562, SU-DHL-1, SU-DHL-4, Daudi, Raji) and the breast cancer cell lines (MDA-MB-468, SK-BR-3). Targets also tended to cluster by protein target type. For example, clustering was observed for: the housekeeping proteins Histone H3 and Ribosomal Protein S6 (RPS6); the T cell markers CD3, CD4, and CD8, and the negative control IgGs. PD1, a target that is not expressed in any of the analyzed cell lines, also clustered with the negative control IgGs, showing antibodies with no specificity resemble the behavior of antibodies on samples lacking the expression of target epitopes.