A | B | C | D | E | F | |
Dissociation Enzyme Mix | Stock | Final | 1 rxn (µl) | 4 rxn + 10% (µl) | 8 rxn + 10% (µl) | |
Liberase TH (mg/ml) | 5 | 1 | 420 | 1848 | 3696 | |
RPMI | - | - | 1680 | 7392 | 14,784 | |
Total Volume (µl) | 2100 | 9240 | 18,480 |
A | B | C | D | E | F | |
Quenching Buffer | Stock | Final | 1 rxn (µl) | 4 rxn + 10% (µl) | 8 rxn + 10% (µl) | |
Nuclease-free water | - | - | 437.5 | 1925 | 3850 | |
Conc. Quench Buffer (10x Genomics) | 8X | 1X | 62.5 | 275 | 550 | |
Total Volume (µl) | 500 | 2200 | 4400 |
A | B | |
temp ON | ||
spin - 20 rpm | 5' 0" | |
loop 3X | ||
spin 20 rpm | 14' 0" | |
spin 1700 rpm | 7" | |
spin 1700 rpm | 1" | |
spin -1700 rpm | 2" | |
spin 1700 rpm | 1" | |
spin 1700 rpm | 4" | |
end loop | ||
end |
A | B | C | D | E | F | |
Hyb Mix (add reagents in order listed) | PN | 1X (µl) | 1X + 20% (µl) | 4X + 20% (µl) | 16X + 20% (µl) | |
Hyb Buffer B | 2000485/ 2000483 | 70 | 84 | 336 | 1344 | |
Enhancer | 2000482 | 10 | 12 | 48 | 192 | |
Total (µl) | 80 | 96 | 384 | 1536 |
A | B | C | |
Step | Temperature | Time | |
Pre-equilibriate | 42C | Hold | |
Probe hybridization | 42C | 24 h* |
A | B | C | D | |
Post-Hyb Wash Buffer (add reagents in order listed) | PN | Pooling 4 samples (mL) | Pooling 16 samples (mL) | |
Nuclease-free water | - | 4.95 | 13.86 | |
Hyb Buffer B | 2000533 | 0.275 | 0.77 | |
Enhancer | 2000482 | 0.275 | 0.77 | |
Total (µl) | 5.5 | 15.40 |
A | B | C | |
Post-Hyb Resuspension Buffer (Add reagents in the order listed) | PN | 1 Pool + 10% (µl) | |
Nuclease-free water | - | 1567.5 | |
Conc. Post-Hyb Buffer | 2000533 | 82.5 | |
Total | 1650.0 |
A | B | |
Starting Total Cell Number in Pool | Post-Hyb Resuspension Buffer (µl) | |
<1 x 10^6 | 550 | |
1 x 10^6 - 4 x 10^6 | 800 | |
5 x 10^6 - 8 x 10^6 | 1050 | |
9 x 10^6 - 12 x 10^6 | 1300 | |
13 x 10^6 - 16 x 10^6 | 1550 |
A | B | C | |
GEM Master Mix (Add reagents in the order listed) | PN | 1X* (µl) | |
GEM Reagent Mix | 2000491 | 20.9 | |
Reducing Agent B | 2000087 | 1.7 | |
GEM Enzyme Mix | 2000490 | 12.4 | |
Total | 35.0 |
A | B | C | |
Step | Temperature | Time | |
1 | 25C | 60 min | |
2 | 60C | 45 min | |
3 | 80C | 20 min | |
Hold | 4C | Hold |