Step 1: Reverse Transcription
The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.
Library generation starts with oligodT priming containing the Illumina-specific Read 2 linker sequence.
Step 2: Removal of RNA
After first strand synthesis the RNA is removed.
Step 3: Second Strand Synthesis
Second strand synthesis is initiated by random priming and a DNA polymerase. The random primer contains the Illumina-specific Read 1 linker sequence.
No purification is required between first and second strand synthesis. Second strand synthesis is followed by a magnetic bead-based purification step rendering the protocol compatible with automation.
Step 4: Library Amplification
During the library amplification step sequences required for cluster generation are introduced.
Multiplexing can be performed with up to 384 barcode combinations using the 96 available i7 indices and four i5 indices.
Step 5: Sequencing
NGS reads are generated towards the poly(A) tail and directly correspond to the mRNA sequence. To pinpoint the exact 3’ end, longer reads may be required (SR100, SR150). Although paired-end sequencing is possible, we do not recommend it for QuantSeq FWD. Read 2 would start with the poly(T) stretch, and as a result of sequencing through the homopolymer stretch, the quality of Read 2 would be very low.