Extended Data Fig. 1: Workflow schematic of SPOTS.

(Step 1) Tissue sections are mounted onto Visium slide, fixed for 10 min, and immunostained (Step 2) with fluorescent and TotalSeq-A antibodies (same clones) for 90 min in the presence of poly-T blocking oligos that prevent non-specific ADTs binding to slide surface. After washes, tissue is scanned under a scanning microscope to capture the tissue structure and fiducial spots (Step 3). The tissue is then permeabilized (Step 4), and RNA diffuses onto the poly-T oligos on the spatial barcodes. Following tissue digestion (Step 5), and template switched RT-PCR (Step 6), gene expression and ADT libraries are generated to preserve associated spatial barcodes and RNA sequence/ADT antibody barcodes (Step 7). Gene expression and ADT libraries are sequenced, and data are integrated using our designated pipeline (Step 8).