A | B | |
Primer name | Primer sequence | |
Oligo-dT | 5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′ | |
TSO | 5′-biotin- AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′ | |
ISPCR primer | 5′-AAGCAGTGGTATCAACGCAGAGT-3′ |
A | B | C | D | E | |
1000µl Droplets | Stock concentration | Final concentration | |||
Reagents | g/mol | M | mM | µl use | |
CM-Dextran Sodium Salt | 162.14 | 1 | 60 | 60 | |
PDDA | 174 | 1 | 10 | 5.8 | |
ng/µl | ng/µl | µl use | |||
RNA | 1243 | 50 | 40.2 | ||
Droplet Buffer | 10mM Tris, 4mM MgCl2, pH 8 | 893.7 |
A | B | C | |
Reagents | Stock concentration | 1x [µl] | |
oligo-dT primer | 10 µM | 1 | |
dNTPs | 10 mM each | 1 | |
nuclease free H2O | 2 | ||
MasterMix Total [µl] | 4 | ||
Assay Total [µl] | 4 |
A | B | C | |
Reagents | Stock concentration | 1x [µl] | |
nuclease free H2O | 0.09 | ||
MgCl2 | 1 M | 0.06 | |
Betaine | 5 M | 2 | |
DTT | 100 mM | 0.5 | |
5xI FS Buffer | 5x | 2 | |
RNAse inhibitor | 40 U/µl | 0.25 | |
Superscript II RT | 200 U/µl | 0.5 | |
TSO | 100 µM | 0.1 | |
MM total [µl] | 5.5 | ||
Assay total [µl] | 9.5 |
A | B | C | |
Cycle [Amount] | Temp [°C] | Time [min] | |
1 | 42 | 90 | |
10 | 50 | 2 | |
42 | 2 | ||
1 | 70 | 15 | |
4 | Hold |
A | B | C | |
Reagents | Stock concentration | 1x [µl] | |
KAPA HiFi HS ReadyMix | 2 x | 12.5 | |
IS PCR primer | 10 µM | 0.25 | |
nuclease free H2O | 2.25 | ||
MM total [µl] | 15 | ||
Assay total [µl] | 24.5 |
A | B | C | |
Cycle [Amount] | Temp [°C] | Time | |
1 | 98 | 3 min | |
12 - 23 | 98 | 20 sec | |
67 | 15 sec | ||
72 | 6 min | ||
1 | 72 | 5 min | |
4 | Hold |
A | B | |
Reagent | 1x (µL) | |
Tagment DNA buffer | 2.5 | |
Amplicon Tagmentation mix (Tn5) | 1.25 | |
MM total (µL) | 3.75 |
A | B | |
Reagent | 1x (µL) | |
tagmentation pre-mix | 3.75 | |
pre-diluted cDNA | 1.25 | |
Assay total [µl] | 5 |
A | B | C | |
Cycle [Amount] | Temp [°C] | Time [min] | |
1 | 55 | 10 | |
1 | 10 | Hold |
A | B | |
Reagents | 1x (µL) | |
Index primer 1 | 1.25 | |
Index primer 2 | 1.25 | |
Nextera PCR Master mix | 3.75 | |
MM total [µl] | 6.25 | |
Assay total [µl] | 12.5 |
A | B | C | |
Cycle [Amount] | Temp [°C] | Time | |
1 | 72 | 3 min | |
1 | 95 | 30 sec | |
12 | 95 | 10 sec | |
55 | 30 sec | ||
72 | 60 sec | ||
1 | 72 | 5 min | |
1 | 10 | hold |