Single-cell research is opening up a revolutionary new avenue for understanding gene expression at the individual cell level, enabling the discovery of cellular diversity and offering profound insights into complex biological systems. This approach is particularly valuable for investigating disease mechanisms, cancer, and immunology.

Cost remains one of the greatest obstacles in single-cell sequencing technology. The Chromium platform (10X Genomics) has significantly reduced the per-cell sequencing cost by enabling the analysis of tens of thousands of single cells in a single run. The Chromium platform operates on a chip with eight wells, each capable of accommodating up to 20,000 cells with GEM-X technology. In cases where samples contain a low number of cells, running a single sample per well would fail to exploit the “high throughput” advantage offered by Chromium. Cell multiplexing is the key to addressing this issue. Multiplex solutions in single-cell research enable the simultaneous analysis of large numbers of cells, thus reducing both cost and time.

Common multiplexing methods include Cell Hashing (which tags and differentiates differentiate cell-derived samples within the same pool). For example, BioLegend TotalSeq™-C is compatible with GEM-X 5’, and TotalSeq™-B works with GEM-X 3’, when used in combination with cell hashing antibodies and CellPlex (10x Genomics, which uses distinct CMO-Cell Multiplex Oligos to tag samples). However, these methods require complex, time-consuming sample preparation processes and are typically only compatible with human or mouse cells. To overcome this challenge, 10X Genomics introduced the On-Chip Multiplex (OCM) solution, enabling multiplexing directly on the chip and eliminating the need for complex sample preparation, thus reducing handling time. With the capability to process multiple samples in a single sequencing run, GEM-X Universal OCM offers a flexible, cost-effective solution that still ensures superior data quality. This technology also reduces the need for large cell inputs, making it highly effective for large-scale studies, particularly in disease mechanism, cancer, and immunology research.

Exceptional Performance of GEM-X Universal OCM:

• Reduced cost per sample: Processes up to 8 samples and up to 40,000 cells per chip.
• Multiplex 4 samples per GEM reaction: Equivalent to 5,000 cells per sample.
• Simplified multiplexing process: No complex or high-cost procedures required.
• Time-efficient: GEM creation on the machine takes approximately 4 minutes.
• High cell recovery: Effective even with small cell inputs.
• Suitable for limited samples: Such as organoids (cell clusters grown in 3D culture) and stem cells.
• Broad analysis scope: Applicable to many species, not limited to humans and mice.

Performance Evaluation:

UMAP and bar charts demonstrate the recovery performance and cell-type distribution similarity between the Singleplex and Multiplex methods when analyzing the same human PBMC sample using the GEM-X 5′ Gene Expression solution. Results show that both methods achieve comparable recovery performance and cell-type proportions, proving the multiplex method’s effectiveness and accuracy in preserving cell-type structure compared to Singleplex.

Cell recovery and cell-type proportions also show high consistency across different lanes in the multiplex method (with each lane recovering approximately 4,500–5,000 cells), ensuring lanes do not interfere with each other, thereby enhancing the accuracy and reliability of the results.

The TCR clonotype pairing and detection rate demonstrates equivalence between GEM-X Multiplex and GEM-X Singleplex methods when analyzing the same human PBMC sample, with 5,000 cells in both GEM-X Singleplex and 5,000 cells per lane in GEM-X Multiplex. T-cell clonotypes are represented in hexagonal plots, visualizing the distribution and diversity of T-cell clonotypes in the analyzed sample.

The clonotype pairing and detection rates between GEM-X Multiplex and GEM-X Singleplex lanes also show consistent, confirming the ability of both methods to evenly and effectively analyze the same sample.

Conclusion:

The GEM-X Universal OCM solution by 10X Genomics represents a significant advancement in single-cell research, enabling the simultaneous processing of multiple samples on a single chip at a cost-effective and efficient rate. Traditional multiplex methods, while effective, require complex and time-consuming sample preparation processes, and are typically only compatible with specific sample types, which can limit scalability and throughput. GEM-X OCM addresses this issue by simplifying the process, reducing costs and time, while maintaining high accuracy and consistency in the analysis. With its flexible processing capability and compatibility with diverse sample types, GEM-X OCM is the ideal tool for research on disease mechanisms, cancer, and immunology, opening new opportunities for large-scale studies and advanced biomedical applications.

References

1. GEM-X Universal 3′ Gene Expression Product Sheet: Enhance discovery with industry-leading sensitivity at single cell resolution, https://www.10xgenomics.com/library/dc728c

• GEM-X Universal 5′ Gene Expression Product Sheet: See the immune system at unmatched resolution with more sensitive immune profiling, https://www.10xgenomics.com/library/4ec27d