FFPE Tissues

1. The tissues are permeabilized to release ligated probe pairs that bind to adjacent capture probes on the slide, allowing for the capture of gene expression information.
2. Pairs of probes specific to each gene in the protein-coding transcriptome are hybridized to their gene target and then ligated to one another.
3. The probe pairs are extended to incorporate complements of the spatial barcodes and sequencing libraries prepared.

Fresh-Frozen Tissues

1. In fresh-frozen tissues, RNA is released by fixing and permeabilizing – which binds to adjacent capture probes to allow capture of gene expression information.
2. Then sequencing libraries are prepared by synthesizing and capturing RNA.

(A) an IF-only image

(B) an IF image overlaid with Visium data containing total UMI counts

(C) an IF image overlaid with Visium data for spatially naïve clustering based on total differentially expressed genes

(D) The top 10 genes that are more highly expressed in Cluster 6 are shown to the right along with example images.

(A) Visium Spatial Gene Expression provides unbiased or targeted gene expression readout in intact sections from fresh-frozen tissue. The workflow is compatible with H&E to provide morphological context and IF staining to co-detect protein from the same tissue section.

(B) Shown on the left is an H&E image for a coronal mouse brain section, followed by an overlay of Visium data for total unique molecular identifiers (UMIs) for whole transcriptome analysis or spatially naïve spot clustering based on total differentially expressed genes. Listed on the far right are the most highly expressed genes in Cluster 4.

(Source: 10x Genomics)

Histological tools like in situ hybridization or immunohistochemistry are limited in the breadth of analysis they can perform. With protein detection by immunofluorescence one can visualize spatial patterns of gene expression.

The Visium spatial gene expression solution increases the level of precision by blending immunofluorescence protein detection and unbiased, spatial gene expression in the same tissue section alongside histological analysis.

The Significant Difference - the ability to localize rare cell subtypes in patient samples, characterize immune cell activation state, or resolve post-translational modifications and cell signaling in intact tissue, without letting go of whole transcriptome data. The combined workflow of Visium Spatial Gene Expression with Immunofluorescence makes it simpler to implement spatial transcriptomics technology into standard methods of tissue sectioning and immunofluorescence staining.

Space Ranger Analysis Pipelines:

• Process Visium data with both brightfield and fluorescence microscope images.
• Easy access to sample demultiplexing, image alignment, barcode processing, gene counting at your fingertips

Loupe Browser Visualization Software:

• Rapidly locate important genes, cell types, and substructure without the need to program