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## Tips & Best Practices |
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Tips & Best Practices section includes additional guidance |
## Emulsion-safe Plastics |
- l Use validated emulsion-safe plastic consumables when handling GEMs as some plastics can destabilize GEMs. |
- l Consult Chromium GEM-X Single Cell Gene Expression v3 & Immune Profiling v4 - Protocol Planner (CG000748) for a detailed list of plastics and other consumables. |
## Cell Concentration |
- l The optimal input cell concentration depends upon the desired cell recovery target: |
- l The presence of dead cells and debris in the suspension may reduce the recovery rate. Consult the following documents for more information on preparing cells: |
- o 10x Genomics Single Cell Protocols Cell Preparation Handbook (Documents CG00053) |
- o Best Practices to Minimize Chromium Next GEM Chip Clogs and Wetting Failure (Document CG000479) |
| Optimal Input Cell Concentration | Cell Recovery Target | |
|------------------------------------|------------------------| |
| 700-1,200 cells/ μ l | 500-10,000 cells | |
| 1,300-1,600 cells/ μ l | 10,000-20,000 cells | |
| Multiplet Rate (%) | # of Cells Loaded | # of Cells Recovered | |
|----------------------|---------------------|------------------------| |
| ~0.20% | ~725 | ~500 | |
| ~0.40% | ~1,450 | ~1,000 | |
| ~0.80% | ~2,900 | ~2,000 | |
| ~1.20% | ~4,350 | ~3,000 | |
| ~1.60% | ~5,800 | ~4,000 | |
| ~2.00% | ~7,250 | ~5,000 | |
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| Multiplet Rate (%) | # of Cells Loaded | # of Cells Recovered | |
|----------------------|---------------------|------------------------| |
| ~2.40% | ~8,700 | ~6,000 | |
| ~2.80% | ~10,150 | ~7,000 | |
| ~3.20% | ~11,600 | ~8,000 | |
| ~3.60% | ~13,050 | ~9,000 | |
| ~4.00% | ~14,500 | ~10,000 | |
| ~4.40% | ~15,950 | ~11,000 | |
| ~4.80% | ~17,400 | ~12,000 | |
| ~5.20% | ~18,850 | ~13,000 | |
| ~5.60% | ~20,300 | ~14,000 | |
| ~6.00% | ~21,750 | ~15,000 | |
| ~6.40% | ~23,200 | ~16,000 | |
| ~6.80% | ~24,650 | ~17,000 | |
| ~7.20% | ~26,100 | ~18,000 | |
| ~7.60% | ~27,550 | ~19,000 | |
| ~8.00% | ~29,000 | ~20,000 | |
## General Reagent Handling |
- l Fully thaw and thoroughly mix reagents before use. |
- l Keep all enzymes and reagent mixes on ice during setup and use. Promptly move reagents back to the recommended storage. |
- l Calculate reagent volumes with 10% excess of 1 reaction values. |
- l Limit Partitioning Oil exposure to air to minimize evaporation. |
- l If using multiple chips, use separate reagent reservoirs for each chip during loading. |
- l Thoroughly mix samples with the beads during bead-based cleanup steps. |
## 50% Glycerol Solution for Addition to Unused Chip Wells |
- l Purchase 50% glycerol solution from Ricca Chemical Company, Glycerin (glycerol), 50% (v/v) Aqueous Solution, PN-3290-32. |
OR |
- l Prepare 50% glycerol solution: |
- o Mix an equal volume of water and ≥99% Glycerol, Molecular Biology Grade. |
- o Filter through a 0.2 μm filter. |
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