PRODUCT | OLIGO SEQUENCE | |
NEBNext Template Switching Oligo | 5´-GCT AAT CAT TGC AAG CAG TGG TAT CAA CGC AGA GTA CAT rGrGrG-3´ | |
NEBNext Single Cell RT Primer Mix | 5´-AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TV-3´ | |
NEBNext Single Cell cDNA PCR Primer | 5´-AAG CAG TGG TAT CAA CGC AGA GT-3´ |
A | B | C | |
COMPONENT | < 5 ng RNA VOLUME (µl) PER RXN | ≥ 5 ng RNA VOLUME (µl) PER RXN | |
Total RNA | Up to 8 µl | Up to 7 µl | |
(lilac) NEBNext Single Cell RT Primer Mix | 1 µl | 2 µl | |
Nuclease-free Water | Variable | Variable | |
Total Volume | 9 µl | 9 µl |
A | B | |
COMPONENT | VOLUME (µl) PER REACTION | |
(lilac) NEBNext Single Cell RT Buffer | 5 µl | |
(lilac) NEBNext Template Switching Oligo | 1 µl | |
(lilac) NEBNext Single Cell RT Enzyme Mix | 2 µl | |
Nuclease-free Water | 3 µl | |
Total Volume | 11 µl |
A | B | |
COMPONENT | VOLUME (µl) PER REACTION | |
(orange) NEBNext Single Cell cDNA PCR Master Mix | 50 µl | |
(orange) NEBNext Single Cell cDNA PCR Primer | 2 µl | |
(white) NEBNext Cell Lysis Buffer (10X) | 0.5 µl | |
Nuclease-free Water | 27.5 µl | |
Total Volume | 80 µl |
A | B | C | D | |
CYCLE STEP | TEMP | TIME | CYCLES | |
Initial Denaturation | 98°C | 45 seconds | 1 | |
Denaturation | 98°C | 10 seconds | 7-21* (See 'Recommended Number of PCR Cycles' table below) | |
Annealing | 62°C | 15 seconds | ||
Extension | 72°C | 3 minutes | ||
Final Extension | 72°C | 5 minutes | 1 | |
Hold | 4°C | ∞ |
A | B | |
TOTAL RNA | RECOMMENDED NUMBER OF PCR CYCLES* | |
2 pg | 20-21 | |
10 pg | 17-18 | |
100 pg | 14-15 | |
1 ng | 10-11 | |
10 ng | 8-9 | |
100 ng/200 ng | 7-8 |
A | B | |
cDNA PCR YIELD | RECOMMENDATION FOR SECTIONS "Fragmentation/End Prep" – "Assess Library Quality and Quantity on a Bioanalyzer" | |
100 pg–1 ng | Use all of the cDNA and adjust PCR cycles (see table in Section ""PCR Enrichment of Adaptor-ligated DNA"") | |
1 ng–20 ng | Typical cDNA yield. Use 8 cycles for the library enrichment PCR. cDNA input into library prep can be normalized. | |
20 ng–100 ng | cDNA input into library prep can be normalized. Adjust PCR cycles per table in Section "PCR Enrichment of Adaptor-ligated DNA". | |
> 100 ng | Normalize cDNA so that at least 4 PCR cycles will be used in the library enrichment PCR (Section "PCR Enrichment of Adaptor-ligated DNA") |
A | B | |
COMPONENT | VOLUME (µl) PER REACTION | |
cDNA (Step 34) | 26 µl | |
(yellow) NEBNext Ultra II FS Reaction Buffer | 7 µl | |
(yellow) NEBNext Ultra II FS Enzyme Mix | 2 µl | |
Total Volume | 35 µl |
A | B | |
COMPONENT | VOLUME (µl) PER REACTION | |
FS Reaction Mixture (Step 40) | 35 µl | |
(red) NEBNext Ultra II Ligation Master Mix | 30 µl | |
(red) NEBNext Ligation Enhancer | 1 µl | |
(red) NEBNext Adaptor for Illumina* (diluted 1:25) | 2.5 µl | |
Total Volume | 68.5 µl |
A | B | |
COMPONENT | VOLUME (µl) PER REACTION | |
Adaptor Ligated DNA Fragments (Step 60) | 15 µl | |
(blue) NEBNext Ultra II Q5 Master Mix | 25 µl | |
(blue) Index Primer/i7 Primer*,** | 5 µl | |
(blue) Universal PCR Primer/i5 Primer*, ** | 5 µl | |
Total Volume | 50 µl |
A | B | |
COMPONENT | VOLUME (µl) PER REACTION | |
Adaptor Ligated DNA Fragments (Step 60) | 15 µl | |
(blue) NEBNext Ultra II Q5 Master Mix | 25 µl | |
Index Primer Mix * | 10 µl | |
Total Volume | 50 µl |
A | B | C | D | |
CYCLE STEP | TEMP | TIME | CYCLES | |
Initial Denaturation | 98°C | 30 seconds | 1 | |
Denaturation | 98°C | 10 seconds | 8* | |
Annealing/ Extension | 65°C | 75 seconds | ||
Final Extension | 65°C | 5 minutes | 1 | |
Hold | 4°C | ∞ |
A | B | |
INPUT IN THE FRAGMENTATION/END PREP REACTION* | # CYCLES REQUIRED | |
100 pg–1 ng | 9–12 | |
1 ng–20 ng | 6–9 | |
20 ng–100 ng | 3–6 |