Universal 3' or 5' Gene Expression
The Universal kits (formerly Next GEM Gene Expression) offer scalable whole transcriptome profiling at the single-cell level, with flexible sample input options. The 3' assay can be paired with cell surface protein staining, while the 5' assay supports full-length TCR/BCR sequencing, antigen specificity, CRISPR guide analysis, and cell surface protein profiling. For cost efficiency, samples can be multiplexed using methods such as Hashtag antibodies, on-chip multiplexing, or genomic variation.
Available Modalities:
Universal 3' Gene Expression from 10X Genomics
Universal 5' Gene Expression from 10X Genomics
Min and max load: 625-25k cells/nuclei
Recovery rate: 80%
On-chip-multiplexing: 5,000 cells/lane, 4 lanes pooled into one reaction
Sequencing depth: 20.000 – 100.000 reads/cell, based on cell type and scientific questions
✅ Tip: It is recommended to perform a pilot experiment with a well-characterized sample and optimize the sample preparation protocol for your specific cell type/tissue.
Sample Types:
- Single Nuclei Suspensions: Fresh cells, Fresh frozen tissue
- Single Cell Suspensions: Fresh tissue, Frozen suspensions, Cell culture
Necessary Quality Requirements:
- 90% single cells/nuclei (microscopy or flow cytometry)
- Viability: >70% for cells (Trypan Blue exclusion)
- Intact nuclear membranes (DAPI staining) for nuclei
Optional (Pilot recommended):
- RIN >8 (Bioanalyzer or TapeStation for fresh cells)
- DV200 >30% for nuclei and methanol-fixed samples
- RT-qPCR for housekeeping genes (e.g., GAPDH, ACTB)
Storage: Methanol fixation is possible for long-term storage (see below).
Resuspension Buffers
- Cells: 1X PBS + 0.04% BSA (standard) or culture media (up to 10% FBS) for fragile cells
- Nuclei: 1X PBS + 1% BSA + 0.2 U/µL RNase Inhibitor
- Methanol fixation: Core staff will rehydrate methanol-fixed samples
Labeling and Storage
- Use low-binding microcentrifuge tubes
- Clearly label tubes/plates with sample names (matching the Excel submission file)
- Keep samples on ice (store up to 30 minutes)
Concentration Guidelines
Target Recovery (cells or nuclei) | Concentration (cells/µL) |
|---|---|
| 500 | 50-100 |
| 5,000 | 100-700 |
| 10,000 | 700–1,200 |
| 20,000 | 1,200–2,500 |
Counting: Conducted by Core on the Cellaca (10µL).
Protocols by 10x Genomics:
Allows simultaneous protein and RNA profiling.
- TotalSeq-A: Separate ADT and HTO library, but with limited support from 10x Genomics.
- TotalSeq-B: Designed specifically for 3' workflows.
- TotalSeq-C: Designed specifically for 5' workflows.
Protocols by BioLegend
✅ Tip: Perform a small-scale test before full processing.
✅ Cell Hashing: Pool samples equally before 10x processing.
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.
Official 10x Genomics protocol
- Methanol fixation may impact some protein epitopes
- Not recommended for CITE-seq applications
- May result in lower gene detection compared to fresh samples
- Core will re-hydrate samples for Controller run
Lead times: 6-8 weeks
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Our Process:
- Single-cell/nuclei suspension quality assessment (optional)
- GEM generation and barcoding using Chromium X
- cDNA amplification (QC)
- Library construction (QC)
- Pooling and Sequencing on NovaSeq X
- Data analysis using Cell Ranger software
- Data delivery through DDS