QuantSeq 3’ mRNA Sequencing Workflow
Step1:
The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.
Step1:
Library generation starts with oligodT priming containing the Illumina-specific Read 2 linker sequence.
Step1:
After first strand synthesis the RNA is removed.
Step1:
Second strand synthesis is initiated by random priming and a DNA polymerase. The random primer contains the Illumina-specific Read 1 linker sequence.
Step1:
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.
Step1:
During the library amplification step sequences required for cluster generation are introduced.
Step1:
Multiplexing can be performed with up to 384 barcode combinations using the 96 available i7 indices and four i5 indices.
Step1:
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.
