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By MedGenome Scientific Affairs
Single-cell RNA sequencing (scRNA-seq) is a powerful method that is widely used in biomedical research. It is extensively used to determine cell composition of complex tissues, identify rare cell types, map heterogeneity at single cell level and identify paired, full-length immunoglobulin sequence and T-cell receptor α/β. Advancements in high-throughput single-cell RNA sequencing technologies, in combination with powerful computational tools, has made scRNA-seq a widely used technology across a broad spectrum of therapeutic areas such as oncology, immunology, neuroscience and developmental biology. Requirement of live cells for most single cell workflows is a bottleneck that limits its wider usage. Advent of 10x genomics Flex protocol has enabled single cell gene expression profiling using fixed samples including FFPE samples. This offers several advantages compared to conventional single cell workflows.
Advantages of using 10x Flex
The conventional methods for scRNA-seq primarily depend on freshly isolated, or cryopreserved cells, rendering them unsuitable for formaldehyde-fixed or FFPE samples. With 10x Genomics’s Chromium Single Cell Gene Expression Flex kit, it is now possible to fix, and store cells or nuclei at -80°C, allowing subsequent analysis without compromising the data quality. Once the fixed single-cell or nuclei samples are prepared for analysis, they undergo hybridization with probe sets designed to target specific regions in the transcriptome. These probe sets are barcoded, facilitating either individual processing in a singleplex or a multiplex workflow. The hybridized transcripts are then amplified to generate sequencing libraries using Gel Bead-in-emulsion (GEM) droplets and the Chromium system. Finally, the samples are sequenced and analyzed using Cell Ranger, a software suite designed specifically by 10x Genomics for performing single-cell RNA sequencing data analysis. An additional multiomic benefit of Flex is the ability to integrate gene expression data with the identification of cell surface proteins at the single-cell level, utilizing both singleplex and multiplex workflows.
A key feature of Flex is its ability to make scRNA-seq adaptable for fragile tissues, ensuring immediate preservation to minimize the loss of quality. Flex is extremely useful when dealing with infectious samples as the samples are fixed. Fixing the samples can neutralize the infectious agents, potentially allowing researchers to handle and analyze samples outside of Biosafety Level 3 facilities, depending on the specific agent, fixation method, and regulatory guidelines. Also, it offers a cost-effective price per cell and is well-suited for large-scale projects. Furthermore, the option for sample multiplexing contributes to decreased batch and experimental variability.
| Features | Flex | 3′ Gene expression |
|---|---|---|
| Type of chemistry | Probe-based | Reverse transcription-based |
| Sample type | Primary cells, dissociated fresh or fixed tissue, including FFPE and cell lines | Primary cells, dissociated fresh tissue and cell lines |
| Cell throughput | Singleplex: 10,000 cells/channel; up to 80,000 cells/chip Multiplex: 128,000 cells/channel; up to 1,024,000 cells/chip | Low: 1,000 cells per channel, with a maximum of 8,000 cells/chip Standard: 10,000 cells per channel, with a maximum capacity of 80,000 cells/chip. High: 20,000 cells per channel, with a total of 320,000 cells/chip. |
| Species compatibility | Human and Mouse | Human, mouse, rat, model organisms, and plants |
| Number of reads per cell | Between 10,000 and 40,000 | Between 30,000 and 80,000 |
| Cell recovery | High | Variable |
| Sensitivity | High | Moderate |
Exploring spatial gene expression using Visium from 10x Genomics
Spatial transcriptomics is another powerful technique for measuring gene expression across a tissue section, thus providing spatial context. Characterizing spatial distribution of different cell types in healthy and disease conditions can provide significant insights. It can also provide valuable insights into biomarker discoveries, and the elucidation of tumor heterogeneity and its dynamic microenvironments.
Since its inception, spatial transcriptomics has been widely used to study tissue architecture and associated expression pattern in various conditions. Spatial technologies can be broadly categorized into two groups: imaging-based and sequencing-based technologies. The major difference between these two approaches lies in how the spatial localization and abundance of mRNA molecules are determined within a tissue section.
Among several platforms available for spatial transcriptomics, Visium from 10x Genomics is one of the most widely methods. It is an in situ capturing method, wherein the transcript is captured within the tissue and subsequently sequenced externally. The Visium workflow consists of slides, imprinted with oligo capture barcoded probes. The tissue sections are placed onto a glass slide, stained, and then imaged. The tissue sections are then permeabilized, decrosslinked and incubated with transcript specific probes. Transcriptomic probes are then transferred to Visium slides that contain capture probes and extended with barcodes. These barcoded probes are then transferred to microfuge tubes to prepare 10x barcoded sequencing library. These libraries are then sequenced using standard short read sequencing technologies like Illumina.
The Visium technology is compatible with fresh frozen and FFPE tissues.
Both Flex and Visium are powerful tools for single-cell gene expression analysis, but they differ in their capabilities and workflow.
Applications of 10x Flex and Visium
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- Oncology: Characterize tumor heterogeneity and tumor microenvironments
- Drug discovery and development: Understand how drugs affect cells at the single-cell level, identify potential drug targets, and predict therapeutic responses
- Immunology: Decipher the immune response at single-cell level, investigate immune cell composition and dynamics within tissues, understand immune response mechanism to diseases or infections
- Neuroscience: Analyze gene expression in specific brain regions to gain insights into neural circuits and brain function
- Developmental Biology: Characterize diverse cell types within complex tissues, identify gene expression patterns crucial for tissue formation and determine the spatiotemporal dynamics
MedGenome sequencing and bioinformatics solutions
MedGenome is a 10x Genomics Certified Service Provider empowering researchers with cutting-edge single-cell sequencing solutions. Our comprehensive bioinformatics solutions enable researchers to interrogate single cell and spatial transcriptomics data to answer questions ranging from cellular heterogeneity to cell type composition to differential gene expression analysis and much more.
Explore MedGenome’s efficient and rapid 10x Flex and Visium solutions for a budget-friendly option. For detailed insights into our multi omics solutions, connect with the MedGenome scientific team at research@medgenome.com.
References
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- Wang S, Sun ST, Zhang XY, Ding HR, Yuan Y, He JJ, Wang MS, Yang B, Li YB (2023). The Evolution of Single-Cell RNA Sequencing Technology and Application: Progress and Perspectives. Int J Mol Sci. 24(3):2943.
- Du, J., Yang, YC., An, ZJ. et al. (2023). Advances in spatial transcriptomics and related data analysis strategies. J Transl Med. 21, 330.
- Williams CG, Lee HJ, Asatsuma T, Vento-Tormo R, Haque A (2022). An introduction to spatial transcriptomics for biomedical research. Genome Med. 14(1):68.
- https://www.10xgenomics.com/products/single-cell-gene-expression-flex
- https://www.10xgenomics.com/blog/the-gold-standard-transformed-taking-single-cell-rna-seq-further-than-ever-before
- https://www.10xgenomics.com/platforms/visium
#Single cell gene expression, #Single cell RNA sequencing, #10x single cell FLEX kit, #Spatial transcriptomics, #Visium
