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The reagents for library generation and the reaction volumes differ between QuantSeq FFPE and QuantSeq V2 protocols. Specifically:
| QuantSeq FFPE | QuantSeq V2 |
First strand cDNA synthesis | RNA input volume up to 7.5µl Oligod(T) with UMIs Oligo dT provided separately Denaturation step 1min at 95°C 10X FS buffer RT reaction 15min
| |
RNA removal | | Only RS enzyme 5µl reaction volume
|
Second Strand Synthesis | 5µl SS1 buffer 15 min incubation 2µl SS2/E2 reaction mix
| 10µl SS1 buffer 30 min incubation 5µl SS2/E2 reaction mix
|
Purification | | |
The overall protocol time remains similar between the two library preps, with a total prep time of 4.5 hours (1.45 hours of hands-on time).