Flex Gene Expression
Flex Gene Expression by 10x Genomics enables high-sensitivity single-cell profiling while preserving sample biology through fixation at collection. This approach stabilizes RNA, allowing flexible handling and batching without compromising data quality. As the most sensitive 10x Genomics application, it supports built-in sample multiplexing, processing up to 16 samples in a single GEM reaction. It also allows analysis of cell surface and intracellular proteins and enables large-scale CRISPR screens.
The provided information applies to the latest GEM-X versions. Older kit versions (formerly Next-GEM) will still be supported until they are discontinued.
Flex Gene Expression from 10X Genomics
Species: Human, mouse, or other (custom probes available)
Sample Fixation: Yes, up to 12 months at -80°C
Capture: Probes targeting 18,000 transcripts
Excluded genes:
- TCR & IG variable/joining regions
- Ribosomal & mitochondrial ribosomal proteins
- Read-through genes
- KIR & HLA genes (high allelic diversity)
- Non-coding RNA
Recovery range: 500 – 20,000 cells/nuclei
Sequencing depth: 20,000 – 500,000 reads/cell (varies by cell type & research question)
⚠️Using <50,000 cells/barcode may limit max cell recovery
✅ Tip: Run a pilot with a well-characterized sample to optimize preparation for your cell type/tissue.
Sample Fixation:
Use only recommended consumables & reagents:
- Blood Fixation and Cell Isolation
- Fixation of Cells & Nuclei
- Tissue Fixation & Dissociation
- Sample Preparation from FFPE Tissue Sections
- Cell Surface & Intracellular Protein Labeling
Quality Requirements:
- Viability:
- >80%: Best sensitivity and recovery.
- ≤50% or lower: Supported, but may reduce sensitivity & increase variability
- Minimize debris to reduce RNA contamination and background noise.
- Filter out aggregates (e.g., 30 µm filters) before fixation.
⚠️Sample quality is the user’s responsibility and directly affects assay performance (viability, aggregates, debris, cell size, etc.).
Labeling and Storage
- Use low-bind microcentrifuge tubes (e.g. Eppendorf)
- Clearly label tubes/plates (match names in the Excel submission file)
- Store long-term at −80ºC
Protocols by 10x Genomics:
- GEM-X Flex Gene Expression Reagent Kit for Singleplex
- GEM-X Flex Gene Expression Reagent Kit for Multiplex samples
- GEM-X Flex Gene Expression Reagent Kit for Singleplex samples with Feature Barcode technology for Protein Expression
- GEM-X Flex Gene Expression Reagent Kit for Multiplex samples with Feature Barcode technology for Protein Expression
Antibody Staining for cell surface/intracellular protein and hashing
Simultaneous Protein & RNA Profiling
- TotalSeq C (BioLegend)
- MultiPro DNA-labeled antibodies (Proteintech)
Protocols
10x Genomics Cell Surface & Intracellular Protein Labeling
General Guidelines by 10x Genomics
- Nozzle: 100 μm and up
- Sheath fluid: no EDTA or excessive magnesium
- Collection Buffer: 5-20% FBS in 1X PBS or 100% FBS for fragile cells
- Resuspending: Centrifuge at 200 rpm for 10 min at 4°C; remove supernatant, 1X PBS with 0.04% BSA
- Precision Sorting: Use a 96-well plate for lower volumes.
- Timing: Process samples within 30 minutes of sorting.
- Viability Staining: Consider DAPI exclusion.
✅ Tip: Do a test run, let samples sit on ice for 30 min and remeasure viability. This will likely reflect the viability at the start of sample processing.
Lead times: 6-8 weeks
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Our Process:
- Fixed Single-cell/nuclei suspension quality assessment
- Probe Hybridization
- Post-Hybridization Washing & Pooling
- GEM generation and barcoding using Chromium X
- Library construction (QC)
- Pooling and Sequencing on NovaSeq X
- Data analysis using Cell Ranger software
- Data delivery through DDS