To generate longer libraries, use the QuantSeq-Flex First Strand Synthesis Module (Cat. No. 166). The protocol is outlined in the QuantSeq-Flex First Strand Synthesis Module User Guide (166UG323).
The figure below shows an example of longer libraries achieved using this protocol.
Figure | Bioanalyzer traces of QuantSeq FWD (std.) and Flex libraries prepared from 500 ng UHRR input RNA. Input RNA was denatured for 3 minutes at 85 °C, with either 5 μl oligodT from the QuantSeq-Flex First Strand Synthesis Module (Cat. No. 026; blue trace, RNA+dT), or the standard QuantSeq FWD FS1 buffer (red trace, std.). Average library size is increased when RNA+dT conditions are used. Libraries were amplified with i7 and i5 6 nt index primers, using Dual PCR Mix (Lexogen i5 6 nt Dual Indexing Add-on Kit, Cat. No. 047) and 13 PCR cycles.