Spatial Transcriptome Sequencing

Spatially patterned gene expression

Spatial Transcriptome Sequencing

Service Overview

Spatial transcriptome sequencing empowers resolving of mRNA profile while retaining information of spatial position. Matching gene expression profile with individual tissue section leads to more comprehensive molecular understanding of tissues, e.g. spatial-specific expression of functional genes, etc.

Spatial Transcriptome Sequencing Workflow


1. A fresh-frozon tissue section is imaged for histological purposes.

2. Tissue section is placed on an array containing RNA-binding probes. Tissue is fixed and permeabilized to release cellular RNA to bind to probes.

3. cDNA is synthesized from captured RNA and prepared for library construction.

4. cDNA library is processed for high-throughput sequencing.

5. Data visualization to determine which genes are expressed, and where, as well as in what quantity.

10X Genomics Spatial Transcriptome

In 10X Genomics, each slides for library construction contains 4 RNA capture regions with area of 6.5 x 6.5 mm2, containing 5000 barcoded spots, i.e. each spot owns a unique barcode. The diameter of each barcoded spot is 55 μm and the center-to-center distance between each two spots is 100 μm.

Cellular mRNA in each tissue section is released and moved into each spot, where mRNA will be ligated to corresponding barcode. These barcoded mRNA is further processed from library construction and sequencing.

Finally, data analysis is based on the barcode information on each reads to trace back where each mRNA is originated, which achieves spatial-specific study on gene expression.

Service Applications

Pathology
Gene expression in morphological context
Immunology
Immune cell infiltrating, spatial pattern of gene expression in immune cells
Developmental Biology
Tissue based studies on morphology related genes.
Neuroscience
Gene expression profile on each section of brain.
Oncology
Tumour microenvironment, tumour infiltrating lymphocytes, etc.

Results Demo






Spatial mRNA expression and clustering of mouse kidney

A. HE staining of tissue section. B. Merging of total UMI and tissue spatial information. C. Merging of total genes and tissue spatial information. D. Spatial clustering of total differentially expressed genes. Top 10 DEGs in cluster 2 is listed on the right. E, F, G, H show spatially expression of four genes.







Spatial resolved gene expression of mouse brain.

A. HE staining of tissue section. B and C show spatially expression of two hippocampus marker genes: Tmsb4x and Selenow. As shown in the figure, a significant higher expression of two genes can be observed in hippocampus region, which meets the expected gene expression pattern.

FAQ

1How to determine sequencing data size?
Sequencing data size mainly depends on sample type, coverage and expression. Normally, we recommend to sequencing ≥ 50 K reads pairs per spot (50% tissue coverage). In this case 50,000 read pairs per spot x 5,000 spots=125 million read pairs in total or 30-60 G per sample.
2How to process sample preparation?
Major experimental process includes: Fresh tissue sampling - Freezing in Isopentane - OCT embedding - cryosectioning - Staining - Imaging with microscopy - Library construction and sequencing. In above procedures, Fresh tissue sampling - Freezing in Isopentane - OCT embedding need to be finished on customer's side. At least three pieces are required for each sample (Minimum 2 for extremely precious material). The size of each piece should not be too big. (Approximate 0.8x0.8x0.8 cm3) Three pieces of samples for: 1) Normal RNA extraction to ensure the sample is good for downstream experiment. (RIN>8); 2)Sectioning and following sequencing; 3) Backup.