10x Genomics Standard Visium Direct Placement

Unbiased molecular profiling is where any species tissue type is placed directly over capture areas that collect whole-transcriptome information at 55-µm spot-based resolution. Since this is a species-agnostic method, frozen tissue is required.

For the 10x Genomics Visium Spatial Gene Expression slides, frozen tissue sections are directly placed into fiducial frames containing about 5,000 barcoded spots in the 6.5 mm-by-6.5 mm capture areas or about 14,000 barcoded spots for the 11 mm-by-11 mm capture areas.

Each 55-µm spot contains millions of oligonucleotides that consist of a:

• 30-nucleotide poly(dT) sequence to capture poly(A) RNA molecules.
• Unique molecular identifier used to identify duplicate molecules arising during library preparation and sequencing.
• Spatial barcode shared by all oligonucleotides within a spot.
• Partial TruSeq Read 1 sequence for use during the library preparation and sequencing workflow.

After tissue is placed onto the Spatial Gene Expression slides:

• Tissue is stained for hematoxylin and eosin or immunofluorescence and imaged.
• Tissue is digested, releasing the RNA molecules that bind to the capture probes on the slide.
• Polymerase chain reaction amplification and library construction steps are performed.
• Libraries are sequenced.

During the Visium workflow, two main data types are captured: a tissue image and sequencing data. 10x Genomics provides two software tools to process and visualize these Visium data types: Space Ranger and Loupe Browser.

Space Ranger processes the input file types to align the Visium sequencing data with the image. Each spatial barcode with the associated unique molecular identifiers captured during the Visium workflow is assigned a spatial location in the tissue image. Space Ranger produces various output files that can be used in Loupe Browser or third-party tools to visualize and analyze spatial data.