With QuantSeq Reverse (REV, Cat. No. 016), the Read 1 linker sequence is located on the 5' end of the oligo(dT) primer and the read 2 linker sequence is located on the 5' end of the random primers used during second strand synthesis. This generates libraries that have a reverse orientation compared to those generated by the QuantSeq FWD kit.
A Custom Sequencing Primer (CSP, included in the kit) is required for sequencing QuantSeq REV libraries, in order to avoid sequencing the poly(T) stretch at the start of read 1. This enables read 1 to begin directly at the exact 3' end of the transcript.
Using Paired-End sequencing is advantageous to identify 3' UTR isoforms.
The use of QuantSeq REV is therefore ideal for applications including alternative polyadenylation site identification and alternative 3' UTR expression studies.