Information about the location of cells in tissues is important for understanding the mechanisms of disease and cancer development. Spatial transcriptomics is a new technological breakthrough that can reveal gene activity in tissues and simultaneously reveal the precise regional location of these active signals. In 2016, various spatial transcriptomic technologies began to be published and have since been successfully applied in the fields of developmental biology, oncology, neuroscience, and neurological diseases.

Advantages of 10x Spatial Transcriptomics

10x Visium technology is one of the most mature spatial transcriptome technologies, and has the following advantages over other technologies:

1. Obtain unbiased high-throughput gene expression: The entire transcriptome can be analyzed in the entire tissue, not just limited to a small region of interest.

2. High spatial resolution: The diameter of each spot is 55 μm, which means that there are 1 to 10 cells on average on each spot, and the number of cells varies slightly depending on the tissue sample.

3. Complete transcriptome gene expression can be obtained from intact tissue, so there is no need to dissociate tissue, and there is no tissue dissociation bias.

4. It can combine clinicopathological information to combine pathological features on tissue with gene expression.

5. Easy-to-use analysis software incorporates histological gene expression data to simplify data analysis.

6, Large detection area (42.5 mm²)

7. High detection efficiency (sensitivity)

8, Complete and simple operation process

9, It can be applied to membrane protein, antigen, immunographic detection, etc.

Principle of 10x Spatial Transcriptomics technology

Frozen tissue sections were placed in the capture area of the 10X Genomics Visium chip. After HE staining and imaging, the tissue sections were permeabilized to release intracellular mRNA, which was captured by the probe with oligo-dT on the chip.  And each probe has a specific address sequence, and then uses mRNA as a template to synthesize cDNA and construct a library, and then to obtain gene expression information through sequencing, and also to obtain the location information of gene expression.

10x Spatial Transcriptomics (10xgenomics.com)

• One slide can hold 4 sections (capture area), prepare 4 libraries;
• 5000 barcoded ST sites per 6.5mm x 6.5mm capture area;
• Millions of UMI probes per ST site;
• The diameter of each spot is 55μm, and the distance between spots is 100μm;
• Each spot covers 1-10 cells and detects thousands of genes;
• Capture RNA with PolyA by Poly T, and use unique spatial barcode for spatial location labeling

10x Spatial Transcriptomics workflow

1. Sections of fresh frozen tissue specimens are placed in the four capture areas of the slide, one section per area.

2. Stain tissue sections on slides using standard fixation and staining techniques, including H&E staining.

3. Tissue permeabilization, releasing mRNA from the cell, which binds to the spatial barcode oligonucleotides on the spot.

4. Reverse transcription to synthesize the second strand of cDNA.

5. The barcoded cDNA is collected for library construction.

6. The constructed library is sequenced.

Tissue Optimization

Tissue permeabilization optimization refers to pre-experiment with tissue permeabilization chip before conducting formal experiments. Because the structural proteins of different tissue types are different, the time required for tissue permeabilization is also different, so it is necessary to explore the optimal time for tissue permeabilization in advance.

Before gene expression library experiments, it is recommended to use the Visium Spatial Tissue Optimization Kit for testing to determine the optimal permeabilization time.

The flow of the tissue experiment is shown below: Tissue optimized slides do not have a spatial barcode and the final result is a fluorescence cDNA footprint image. We select the optimal permeabilization time based on the fluorescence cDNA footprint image.

For 10x spatial transcriptome libraries, Illumina Hiseq PE150 or NovaSeq PE150 sequencing strategy is recommended to measure 50,000-100,000 reads per spot, i.e. 75-100G data volume per sample (5000 spots). The sequencing depth is related to the coverage area of the sample capture area on the glass slide and the type and complexity of the sample and the detection rate of genes can be improved by increasing the sequencing data volume.

Tissue optimization differs from the general library building step in two ways.

① The dNTP with Cy3 fluorophore used in reverse transcription is used to assess tissue permeabilization;

②Because the autofluorescence of the tissue will interfere with fluorescence imaging, the tissue needs to be removed before imaging.

The optimal tissue permeabilization time is determined by three conditions:

① The fluorescence signal is the strongest;

② The fluorescence signal diffusion is the weakest;

③ Select a longer tissue permeabilization time when the first two conditions are consistent.

10x Spatial Transcriptomics Analysis Worklow

After obtaining Sequenced Reads, bioinformatic analysis is performed by the following procedure. The software Space Ranger is used to combine the gene expression information with the location information of the tissue sections to visualize the spatial distribution of cell subpopulations and cell subsets on the tissue sections and the spatial distribution of differential genes. Subsequent functional enrichment of these differential genes is performed to identify the function of cell subsets. In addition to the results of Space Ranger, we also provide basic analysis results of the third-party software Seurat. In addition, we can also provide cell definition, SNV analysis, CNV analysis, ligand-receptor analysis, etc. according to customer requirements.