With QuantSeq Reverse (REV, Cat. No. 225 016), the Read 1 linker adapter sequence is located on the 5' end of the oligo(dT) primer and the Read 2 linker adapter 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.
Figure | Schematic overview of the QuantSeq REV V2 library preparation workflow (Cat. No. 225). Sequencing
read orientation for QuantSeq REV is depicted, with Read 1 (green) and Read 2 (blue). Read 1 reflects the cDNA sequence. QuantSeq REV is suitable for paired-end sequencing, and Read 2 reflects the mRNA sequence. A Custom Sequencing Primer (CSP Version 5, included in the kit) is required for Read 1.
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.