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. Author manuscript; available in PMC: 2023 Dec 1.
Published in final edited form as: Nat Biotechnol. 2023 Jan 2;41(6):788–793. doi: 10.1038/s41587-022-01536-3

Figure 1: Spatial PrOtein and Transcriptome Sequencing (SPOTS).

Figure 1:

(a) Overview of SPOTS workflow outline. Fresh tissue samples were collected and embedded in OCT. Tissue sections (10μm) were processed following SPOTS protocol (including staining with fluorescent and ADT antibodies, permeabilization, mRNA capture, reverse transcription, and second-strand synthesis; Methods) and sequenced for downstream analysis. See Supplementary Figure 1 for detailed SPOTS schematic. Scale bar 200μm.

(b) Normalized ADT levels of key surface markers for mouse spleen. Left panel: immunofluorescence (IF) staining for CD29 (green) and CD4 (red) with inset showing the germinal center (GC) architecture and its underlying spatial barcodes. Right panel: normalized ADT levels of marker genes for T cells (CD3, CD4, CD8), B cells (CD19, B220, IgD), and Macrophages (F4-80, CD163, CD68).

(c) Correlation between the 21 ADTs and their corresponding mRNA expression levels across two biological replicates (Supplementary Table 1).

(d) Spatial barcode clustering and major cell type enrichment (Macrophage, B cell, and T cell) based on ADTs of two biological replicates (Supplementary Table 2).

(e) ADT and mRNA signatures for each cluster of spatial barcodes. Left panel: heatmap showing expression levels (Z-score) of differentially expressed ADTs for each cluster. Right panel: heatmap showing expression levels (Z-score) of differentially expressed mRNAs in 3,000 downsampled spatial barcodes. Key marker genes are highlighted (Supplementary Table 3).

(f) Physical distance (color scale) from the center of GCs for each spatial barcode.

(g) Spatial gene expression patterns of ADTs in the spleen. ADT Expression pattern (Z-score) from GC to spleen cortex and their anatomical definition (black boxes). The top color bar represents the physical distance as in the right panel. The number of spatial barcodes in each bin is labeled at the bottom. MZ, marginal zone.

(h) Cell-type deconvolution based on ADTs expression (Z-score) of each spatial barcode overlaid onto the spleen tissue. Deconvolution values are provided in Supplementary Table 2.