Description

Spatial transcriptomics is a technology that analyzes RNA-seq data from a spatial perspective, enabling the profiling of all mRNA in individual tissue sections. This allows for the localization and differentiation of functional gene expression within specific regions of a tissue. The physical location of cells within a tissue is a key factor determining their molecular characteristics.

• Bulk Transcriptomics: Provides information on the overall expression of mixed cells in a tissue, but does not resolve cellular expression heterogeneity, meaning it cannot provide expression details for individual cells.
• Single-Cell Transcriptomics: Technologies like those from 10x Genomics enable the profiling of gene expression at the individual cell level, thereby resolving cellular heterogeneity within a tissue.
• Spatial Transcriptomics: Technologies such as 10x Visium combine tissue sectioning with transcriptomics sequencing to obtain both spatial and transcriptomic information. This adds spatial distribution data of gene expression, further enhancing the resolution within the tissue.

Principles

1. Tissue Sectioning and Imaging
   Slice fresh frozen tissue samples and perform imaging.

2. Probe Hybridization and Fixation
   Place the tissue sections on slides containing RNA-binding capture probes, followed by fixation and permeabilization (to release mRNA from cells and bind it to the corresponding capture probes to obtain gene expression information).

3. cDNA Synthesis and Library Preparation
   Use the captured RNA as a template to synthesize cDNA and prepare sequencing libraries.

4. Sequencing
   Perform sequencing on the prepared libraries.

5. Data Visualization and Analysis
   Analyze and visualize the data to determine which genes are expressed, the levels of gene expression, and the spatial locations of these genes.

10x Genomics Spatial Transcriptomics Sequencing Technology Principle

In 10x Genomics Spatial Transcriptomics, each slide used for library construction has four capture areas, each measuring 6.5×6.5 mm and containing 5,000 barcoded spots. Each spot has a diameter of 55 μm, and the distance between the centers of adjacent spots is 100 μm. Each spot is assigned a unique barcode sequence.

mRNA is released from cells in the tissue section and migrates to each spot, where it is tagged with the corresponding barcode sequence. The library is then constructed and sequenced.

Finally, the data is analyzed based on barcode information to determine the location of each data point, enabling visualization of spatial gene expression.

Applications

• Pathology: Draw morphological conclusions by adding the dimension of gene expression.
• Immunology: Analyze immune cell infiltration and gene expression features within immune cells.
• Developmental Biology: Identify genes related to morphological development within the tissue context.
• Neuroscience: Map various cellular layers of the brain and differentiate between normal and diseased brain anatomy.
• Oncology: Study tumor microenvironment, tumor heterogeneity, disease progression, cancer morphology, and tumor-infiltrating lymphocytes.