Step 1: Reverse Transcription

time_quantseq01

 

 

The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.

time_quantseq02

 

 

Library generation starts with oligodT priming containing the Illumina-specific Read 2 linker sequence.

Step 2: Removal of RNA

time_quantseq03

 

 

After first strand synthesis the RNA is removed.

Step 3: Second Strand Synthesis

time_quantseq04

 

 

Second strand synthesis is initiated by random priming and a DNA polymerase. The random primer contains the Illumina-specific Read 1 linker sequence.

time_quantseq05

 

 

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

06quantseq_workflow_upd

time_quantseq06

 

 

During the library amplification step sequences required for cluster generation are introduced.

07quantseq_workflow_upd

time_quantseq06

 

 

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.