As your research continuously evolves, so should your methods. Unlock the full potential of single-cell technology with the latest innovations in transcriptomic profiling from 10x Genomics. The Chromium GEM-X Single Cell 3’ Gene Expression v4 and GEM-X Single Cell 5’ Immune Profiling v3 assays are designed to maximise biological insights across fields like cancer, neuroscience and immunology. These more advanced assays are powered by GEM-X technology, which provides a robust foundation for expanding innovation and application support, allowing you to plan for the future of your research.
Another relatively new assay in the 10x Genomics single-cell toolkit is the Chromium Gene Expression Flex assay, also known as the Fixed RNA Profiling assay. The Gene Expression Flex assay represents a major advancement in single-cell transcriptomics, providing researchers with the flexibility to work with fresh, frozen, and fixed samples—even FFPE tissue. By employing a probe-based chemistry, Flex enables high-resolution detection of gene expression across a wide range of cell types, making it ideal for large-scale studies and multi-batch experiments.
When comparing the GEM-X 3′ and 5′ assays with the Gene Expression Flex assay, it’s essential to understand their respective strengths and applications to ensure you select the right assay for your single cell project. In the following sections we will expand on the differences between these three assays, highlighting key factors such as chemistry, sensitivity, application flexibility, and throughput. By understanding these distinctions, you’ll be better equipped to choose the assay that best aligns with your research goals.
GEM-X 3′ and 5′ Assays:
The GEM-X technology builds on the Chromium single-cell platform, offering enhanced performance for both 3′ gene expression and 5′ immune profiling assays compared to the previous Next GEM chemistry. GEM-X is designed to improve cell partitioning, throughput, and reduce multiplet rates, all of which are essential for high-quality single-cell sequencing experiments.
Updated Kit versions:
- GEM-X Single Cell 3’ Gene Expression v4 (also known as the 3’ assay)
- Whole transcriptome gene expression analysis.
- Supports detection of Cell Surface Proteins using TotalSeq-B antibodies.
- GEM-X Single Cell 5’ Immune Profiling v3 (also known as the 5’ assay)
- Whole transcriptome gene expression analysis.
- Analyse full-length V(D)J sequences for paired B-cell or T-cell receptors.
- Supports detection of Cell Surface Proteins using TotalSeq-C antibodies.
Whole transcriptome analysis of single cells can be performed using either the 3’ or 5’ assays. The most significant difference between these assays is which end of the transcript is captured onto the gel beads. Both solutions use polydT primers for reverse transcription, although in the 3′ assay the polydT sequence is located on the gel bead oligo, while in the 5′ assay the polydT is supplied as an RT primer. A template switching oligo (TSO) is used in both workflows to reverse transcribe the full-length mRNA transcript.
While the 3′ assay focuses on capturing the poly-A tail of mRNA to provide a snapshot of gene expression profiles for each cell, the 5′ assay offers additional capabilities such as B and T cell receptor profiling, making it ideal for immunology research. Both assays support the detection of Cell Surface Protein (CSP) expression by labelling cells with specific oligo-tagged antibodies upstream of the cell partitioning reaction. These oligos will be captured onto the gel beads and sequenced for each cell, giving insights into the levels of protein expression at the cellular level.
Both assays are built on the latest GEM-X microfluidic chips (Figure 1), which offer faster processing times and are optimised for greater reproducibility across multiple runs. GEM-X also allows for Feature Barcoding, providing more options for multi-omic analyses such as combining gene expression data with protein expression or CRISPR guide capture.
GEM-X Chip Features:
Here we highlight the key advantages of GEM-X compared to Next GEM technology:
- Increased sensitivity enabled by a two-fold increase in detected genes and improved capture of rare transcripts, fragile cells, and cells with low RNA content.
- Higher throughput with a two-fold increase in cells captured per channel (up to 20,000 cells per channel).
- More cost effective with a more than 50% reduction in cost per cell.
- Improved sample recovery—up to 80% cell recovery efficiency.
- Enhanced data quality and robustness due, in part, to a halved multiplet rate (0.4% per 1,000 cells) and more efficient GEM generation with no increase in cell stress.
- Faster run times—tens of thousands of cells are partitioned and barcoded in just 6 minutes.
Enhanced sensitivity with GEM-X:
Single-cell RNA sequencing allows researchers to uncover rare transcripts, cell states, and cell populations that other methods, such as bulk RNA-seq and flow cytometry cannot detect. This single-cell resolution opens the doors to discovering previously unknown biology. However, analysing complex samples and identifying rare cells requires sensitive methods capable of detecting lowly expressed transcripts.
In comparative experiments, Chromium GEM-X Single Cell 3’ Gene Expression v4 outperformed its predecessor, detecting 98% more genes and 249% more transcripts per cell in mouse brain nuclei samples compared to Next GEM 3’ v3.1 (Figure 2). Similarly, GEM-X Single Cell 5’ Immune Profiling v3 showed a 61% increase in gene detection over Next GEM 5’ v2 when analysing human PBMCs.
This enhanced sensitivity is particularly important for detecting cells in transient states during differentiation, where gene expression shifts rapidly, and traditional methods struggle to capture these changes. For instance, during the transition from a neural progenitor cell to a neuron, surface markers may not change significantly, making these dynamic states undetectable with flow cytometry. With scRNA-seq’s increased sensitivity, researchers can analyse these subtle transitions, advancing our understanding of developmental biology, aging, and enhancing cell therapy strategies.
Gene Expression Flex Assay
The Gene Expression Flex assay (Flex), uses a probe-based chemistry to capture mRNA, differing from the reverse transcription-based approach of the 3′ and 5′ assays (Figure 3). This assay excels in its flexibility, particularly with fixed samples, and offers a high degree of comparability with the 3′ assay. Flex is compatible with fresh or frozen tissue, FFPE tissue, and single cell or nuclei suspensions, enabling a wide variety of sample types to be used as input. It is highly sensitive, allowing researchers to detect low-expressed genes that may be missed by other methods.
Moreover, the Flex assay demonstrates similar gene expression patterns across cell populations as seen in the 3′ assay, making it an excellent tool for large-scale studies where sample preservation or batch processing is critical. Due to the species-specific probes used in this workflow, Flex is currently only compatible with human and mouse samples, targeting the exons of over 18,000 human genes and 19,000 mouse genes.
Upstream of cell partitioning on the Chromium X-series instrument, samples are first fixed in 4% paraformaldehyde (PFA), which enables storage or transport before processing. This lets users fix fresh samples at the point of collection to lock in biological states and preserve fragile cells until they are ready to be analysed (Figure 4).
Post-Fixation: Samples can be stored short-term at 4°C for up to one week; or long-term at -80°C for up to 6 months.
Post-Hybridisation: Samples can be stored after hybridisation for up to 6 months at -80°C.
After fixation, cells are permeabilised and individual samples are then hybridised to species-specific probe sets, which bind to their respective mRNA targets released from the cell. Samples may be processed individually (singleplex workflow) or pooled with up to 16 samples in a single lane of a Chromium chip (multiplex workflow). During GEM generation, the probe sets are ligated and extended to incorporate unique barcodes. It is the gene-specific probes which are captured onto the gel beads and then eventually sequenced to provide whole transcriptome gene expression analysis of each cell.
Multiplexing capabilities:
While the fixation of samples in the Flex workflow allows for sample storage and batching upstream of running on the Chromium, the use of probes containing unique sample-specific barcodes allows users to multiplex up to 4 or 16 samples per lane on the Chromium chip, with no additional kits or reagents required:
Maximum loading for 4-plex: 40,000 cells/lane or 10,000 cells/barcode.
Maximum loading for 16-plex: 128,000 cells/lane or 8,000 cells/barcode.
In designing an experiment, you therefore have control over three axes – the number of samples you multiplex together, and from that pool the number of cells you add per lane, and finally how many lanes you load. Running the assay with the 16-plex format means that users can maximise their sample throughput: up to 128 unique samples and up to 1 million cells per chip. (8 lanes, 16 samples per lane, 8000 cells per sample). Multiplexing samples into a single lane on the Chromium chip means that less GEM reagents are used, in the case of a 16-plex run only a single reaction of the GEM reagents are needed to process 16 individual samples. This leads to a lower overall cost per sample and makes single-cell analysis accessible to smaller labs with budget constraints.
Flex is a highly sensitive assay, and therefore you can substantially reduce your sequencing costs. High performance is achieved with as few as 10,000 read pairs per cell, this is half the reads required compared to the standard Gene Expression assay. High sensitivity also enables detection of lowly expressed targets such as transcription factors and rare cell sub-types.
These built-in benefits of the Flex assay not only increase the number of cells per sample/channel, but also provide increased sample handling flexibility to enable transportation and storage, reducing batch effects and relieving constraints of running single cell experiments.
Table 1 and the summary section below provide a detailed comparison of the key differences and features between each of the new GEM-X assays and Flex.
| Flex | GEM-X 3’ | GEM-X 5’ | |
|---|---|---|---|
| Species | Human and mouse only | Species agnostic | Species agnostic |
| Gene expression readout | Probe-based detection method | Full length transcript - profile isoforms, lncRNAs, expressed SNPs | Full length transcript - profile isoforms, lncRNAs, expressed SNPs |
| Multiomic capabilities | Cell surface protein only Intracellular protein CRISPR screening | Cell surface protein only* | Cell surface protein BCR/TCR sequencing** CRISPR screening |
| Sample preservation method | Fresh, frozen, fixed (even FFPE) | Fresh, frozen, or fixed | Fresh, frozen, or fixed |
| Study logistics | Sample fixation & batching can ease logistical constraints | If logistics allow for fresh/frozen sample processing | If logistics allow for fresh/frozen sample processing |
| Sample input quantity | ≥300k cells recommended for fixation (100k cells feasible) | Lower cell requirements | Lower cell requirements |
| Sample throughput | Standard: up to 8 samples/chip Multiplexed (built-in): up to 128 samples/chip | Standard: up to 8 samples/chip | Standard: up to 8 samples/chip |
| Cell throughput | Up to 10,000 cells/sample | Up to 20,000 cells/sample | Up to 20,000 cells/sample |
| Cost | Lower cost per cell and sample | - | - |
Summary of Key Differences:
Chemistry: GEM-X assays use reverse transcription, while Flex relies on probe-based chemistry.
Application: GEM-X 5′ supports immune profiling, while Flex focuses on flexibility and fixed samples.
Sensitivity: Flex has comparable or higher sensitivity for certain genes compared to the GEM-X assays, especially with low-expressed genes.
Throughput: GEM-X’s advanced microfluidics offer higher throughput and lower multiplet rates.
Multiplexing: Both technologies have unique strengths depending on your research needs. GEM-X is ideal for high-throughput single-cell experiments, while Flex offers flexibility and sensitivity for studies involving fixed samples or long-term batch processing.
NB: Both the new GEM-X assays, as well as the Gene Expression Flex assay are only compatible with the Chromium X-series of instruments (X, iX, Xo), and cannot be run on the Chromium Controller.
If you are designing a single-cell experiment and need assistance deciding which assay best suits your project requirements, please don’t hesitate to reach out to us at Diagnostech for guidance or more information.
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