To generate longer libraries, use the QuantSeq-Flex First Strand Synthesis Module (Cat. No. 026).
Briefly, mix 5 μl of RNA with 5 μl of the oligodT primer provided 166). The protocol is outlined in the QuantSeq-Flex First Strand Synthesis Module , and denature for 3 minutes at 85 °C, then cool the samples to hold at 42 °C (leave them on the thermocycler). Prepare a mastermix of FS1x 5 μl, FS2x 4.5 μl, and E1 0.5 μl per sample, and pre-warm at 42 °C for 2-3 minutes. Then add the pre-warmed mastermix to the RNA / oligodT samples while keeping the samples on the thermocycler incubating at 42 °C. Mix and spin down quickly then return the samples to the thermocycler to incubate for 15 minutes at 42 °C. Changing the denaturing conditions extends the library insert size (Figure 5).
More information can be found in the QuantSeq 3'mRNA-Seq FWD User Guide, Appendix H: Modulating Insert Sizes, p.30.Have to determine what to do with this FAQ. User Guide (166UG323).
The figure below shows an example of longer libraries achieved using this protocol.