Single-cell sequencing is changing our understanding of fundamental biological processes, including development, immunity, pathology, and disease. Single-cell analysis is now necessary for any research aimed at examining cell populations, their distinct genomic composition, and their responses to treatment. Thus, a more robust, automated system is an urgent need for investigators at UTHSA.
10X Genomics
10X Genomics Chromium platform, application-specific sequencing preparation kits, GSF’s variety of Illumina platforms (NovaSeq 6000 and NextSeq 2000), and their analysis software provide users with the latest, most advanced high-throughput single-cell genomics technology in a fully integrated solution. This new and promising technology enables the generation of excellent genomic data: (1) The Chromium Single Cell Gene Expression Solution provides a comprehensive, scalable solution for cell characterization and gene expression profiling of hundreds to tens of thousands of cells. The latest improvements allow you to detect even more unique transcripts per cell, and with the addition of Feature Barcoding technology, you can get a more complete molecular readout cell by cell—identify cell-specific CRISPR-mediated perturbations or simultaneously measure gene and cell surface protein expression in the same cell, with virtually unlimited possibilities for the additional feature types. (2) The Chromium Single Cell Multiome ATAC and Gene Expression: Simultaneously profile gene expression and open chromatin from the same cell. (3) The Chromium Single-Cell Immune Profiling Solution is a comprehensive approach to simultaneously examining the cellular context of the adaptive immune response and immune repertoires of hundreds to tens of thousands of T and B cells in humans or mice on a cell-by-cell basis.
The GSF performs single-cell analysis through a 10X Genomics Chromium X system. For 10X Genomics single-cell studies, the GSF provides the library preparation and sequencing for the following applications:
- Single-cell RNA-seq: The V3 kit provides 3’ digital gene expression by profiling 500 to 10,000 individual cells per sample; the new V4 kit can profile up to 20,000 individual cells per sample, with a cell recovery rate of up to 80%.
- Single-cell Multiome ATAC and Gene Expression: Simultaneously profile gene expression and open chromatin from the same cell
- Single-cell Immune Profiling: Sequence VDJ and TCR regions while assessing the gene expression profiles of immune cells
- Single-cell gene expression Flex: Allow users to uncover critical cellular insights from fresh, frozen, and fixed samples—even FFPE, providing the ultimate workflow flexibility, letting you fix, batch, and run samples on your own schedule.
- Single-cell ATAC-seq: Analyze thousands of unique open chromatin fragments per cell.
- More applications will follow from the updates of 10X Genomics new products
Some facts you should know before getting started on the single-cell project:
- 10x Genomics’ single-cell technology, the Chromium™ Single Cell, offers multiple options for single-cell analysis:
- Single-cell gene expression Flex is the most sensitive whole transcriptome analysis with the flexibility to fix, batch, and run samples on your schedule.
- Single-cell 3’ gene expression, a versatile solution providing 3′ transcriptomic profiling to explore cellular heterogeneity, novel targets, and biomarkers.
- Single-cell 5’ gene expression with immune profiling for comprehensive analysis of immune diversity, including BCR or TC”R repertoire profiling.
- Single-cell multiome simultaneously profiles the transcriptome (gene expression) and epigenome (ATAC for chromatin accessibility) at single-cell resolution.
- Single-cell ATAC-seq: analyze thousands of unique open chromatin fragments per cell.
Cell preparation is essential:
- 10x Genomics Single Cell protocols require suspending viable single cells or nuclei as input. Minimizing the presence of cellular aggregates, dead cells, noncellular nucleic acids, and potential biochemical inhibitors of reverse transcription is critical to obtaining high-quality data. Please check the 10x Genomics website for cell preparation protocol, which describes best practices to help maintain cell viability and maximize sample quality during sample preparation. General protocols for sample handling, purifying, and counting for both abundant and limited cell suspensions are also provided here: https://www.10xgenomics.com/support/universal-three-prime-gene-expression/documentation/steps/sample-prep/single-cell-protocols-cell-preparation-guide. The general protocols described here are expected to be compatible with many, but not all, cell and sample types. Additional optimization may be required for sensitive samples and solid tissues. For additional information on preparing specific sample types, consult the Demonstrated Protocols available on the 10x Genomics Support website.
- For living cells: The optimal cell density of your single-cell suspension stock is 700-1500 cells/ul, and viability > 90%.
- Please note: 10X single-cell service is by appointment only; we require advance notice due to the nature of these preparations, as we will need someone on standby to receive the cells. Please read 10x Genomics GSF Recommendations before you prepare/submit your samples to the GSF.
Please check single-cell submission guidelines (DOC) to see if you are interested in this technology, sample preparation, and data analysis.
If you are interested in working with the GSF for the single-cell experiment, please read Submission of Samples.