Reagent | 1X (ul) | 2X (ul) | 3X (ul) | 4X (ul) | 5X (ul) | 6X (ul) | 7X (ul) | 8X (ul) | |
RT Reagent B | 18.8 | 41.4 | 62.0 | 82.7 | 103.4 | 124.1 | 144.8 | 165.4 | |
Template Switch Oligo | 2.4 | 5.3 | 7.9 | 10.6 | 13.2 | 15.8 | 18.5 | 21.1 | |
Reducing Agent B | 2.0 | 4.4 | 6.6 | 8.8 | 11.0 | 13.2 | 15.4 | 17.6 | |
RT Enzyme C | 8.7 | 19.1 | 28.7 | 38.3 | 47.9 | 57.4 | 67.0 | 76.6 | |
Total | 31.9 | 70.2 | 105.3 | 140.4 | 175.5 | 210.5 | 245.6 | 280.7 |
Lid Temperature | Reaction Volume | Run time | |
53°C | 125 ul | ~55 min | |
Step | Temperature | Time | |
1 | 53°C | 0:45:00 | |
2 | 85°C | 0:05:00 | |
3 | 4°C | Hold |
Reagent | 1X (ul) | 2X (ul) | 3X (ul) | 4X (ul) | 5X (ul) | 6X (ul) | 7X (ul) | 8X (ul) | |
Cleanup buffer | 182 | 400 | 601 | 801 | 1001 | 1201 | 1401 | 1602 | |
Dynabeads MyOne SILANE | 8 | 18 | 26 | 35 | 44 | 53 | 62 | 70 | |
Reducing Agent B | 5 | 11 | 17 | 22 | 28 | 33 | 39 | 44 | |
Nuclease-free Water | 5 | 11 | 17 | 22 | 28 | 33 | 39 | 44 | |
Total | 200 | 440 | 660 | 880 | 1100 | 1320 | 1540 | 1760 |
Reagent | 2X (ul) | 4X (ul) | 6X (ul) | 8X (ul) | |
Buffer EB | 98 | 196 | 294 | 392 | |
10% Tween 20 | 1 | 2 | 3 | 4 | |
Reducing Agent B | 1 | 2 | 3 | 4 | |
Total | 100 | 200 | 300 | 400 |
Reagent | 1X (ul) | 2X (ul) | 3X (ul) | 4X (ul) | 5X (ul) | 6X (ul) | 7X (ul) | 8X (ul) | |
Amp Mix | 50 | 110 | 165 | 220 | 275 | 330 | 385 | 440 | |
cDNA Primers | 15 | 33 | 50 | 66 | 83 | 99 | 116 | 132 | |
Total | 65 | 143 | 215 | 286 | 358 | 429 | 501 | 572 |
Lid Temperature | Reaction Volume | Run time | |
105°C | 100 ul | ~30-45 min | |
Step | Temperature | Time | |
1 | 98°C | 0:03:00 | |
2 | 98°C | 0:00:15 | |
3 | 63°C | 0:00:20 | |
4 | 72°C | 0:01:00 | |
5 | 11 Cycles * | ||
6 | 72°C | 0:01:00 | |
7 | 4°C | Hold | |
* Targeted cell recovery >6,000 |
Lid Temperature | Reaction Volume | Run time | |
65°C | 50 ul | ~35 min | |
Step | Temperature | Time | |
Pre-cool block | 4°C | Hold | |
Fragmentation | 32°C | 0:05:00 | |
End Repair & A-tailing | 65°C | 0:30:00 | |
Hold | 4°C | Hold |
Reagent | 1X (ul) | 2X (ul) | 3X (ul) | 4X (ul) | 5X (ul) | 6X (ul) | 7X (ul) | 8X (ul) | |
Fragmentation Buffer | 5 | 11 | 17 | 22 | 28 | 33 | 39 | 44 | |
Fragmentation Buffer | 10 | 22 | 33 | 44 | 55 | 66 | 77 | 88 | |
Total | 15 | 33 | 50 | 66 | 83 | 99 | 116 | 132 |
A | B | C | D | E | F | G | H | I | |
Reagent | 1X (ul) | 2X (ul) | 3X (ul) | 4X (ul) | 5X (ul) | 6X (ul) | 7X (ul) | 8X (ul) | |
Ligation Buffer | 20 | 44 | 66 | 88 | 110 | 132 | 154 | 176 | |
b) | Add 50 µl Adaptor Ligation Mix to 50 µl sample. Pipette mix 15x (pipette set to 90 µl). Centrifuge briefly | 22 | 33 | 44 | 55 | 66 | 77 | 88 | |
Adaptor Oligos | 20 | 44 | 66 | 88 | 110 | 132 | 154 | 176 | |
Total | 50 | 110 | 165 | 220 | 275 | 330 | 385 | 440 |
Lid Temperature | Reaction Volume | Run time | |
30°C | 100 ul | ~15 min | |
Step | Temperature | Time | |
1 | 20°C | 0:15:00 | |
2 | 4°C | Hold |
Lid Temperature | Reaction Volume | Run time | |
105°C | 100 ul | ~ 25-40 min | |
Step | Temperature | Time | |
1 | 98°C | 0:00:45 | |
2 | 98°C | 0:00:20 | |
3 | 54°C | 0:00:30 | |
4 | 72°C | 0:00:20 | |
5 | # Cycles * | ||
6 | 72°C | 0:01:00 | |
7 | 4°C | Hold |
Sequencing Depth | Total Cycles | |
Sequencing Depth | 14 - 16 | |
Sequencing Depth | 12 - 14 | |
Sequencing Depth | 10 - 12 | |
Sequencing Depth | 8 - 10 | |
Sequencing Depth | 6 - 8 | |
Sequencing Depth | 5 |
A | B | |
Sequencing Depth | Minimum 20,000 read pairs per cell | |
Sequencing Type | Paired-end, dual indexing | |
Sequencing Read | Recommended Number of Cycles | |
Read 1 | 28 cycles | |
i7 index | 10 cycles | |
i5 index | 10 cycles | |
Read 2 | 90 cycles |