Step 1: Reverse Transcription
![01quantseq_workflow 01quantseq_workflow](https://www.lexogen.com/wp-content/uploads/2015/04/01quantseq_workflow.jpg)
The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.
![02quantseq_workflow_rev 02quantseq_workflow_rev](https://www.lexogen.com/wp-content/uploads/2017/02/02quantseq_workflow_rev.jpg)
Library generation starts with oligodT priming containing the Illumina-specific Read 1 linker sequence.
Step 2: Removal of RNA
![03quantseq_workflow_rev 03quantseq_workflow_rev](https://www.lexogen.com/wp-content/uploads/2017/02/03quantseq_workflow_rev.jpg)
After first strand synthesis the RNA is removed.
Step 3: Second Strand Synthesis
![04quantseq_workflow_rev 04quantseq_workflow_rev](https://www.lexogen.com/wp-content/uploads/2017/02/04quantseq_workflow_rev.jpg)
Second strand synthesis is initiated by random priming and a DNA polymerase. The random primer contains the Illumina-specific Read 2 linker sequence.
![05quantseq_workflow_rev 05quantseq_workflow_rev](https://www.lexogen.com/wp-content/uploads/2017/02/05quantseq_workflow_rev.jpg)
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_rev 06quantseq_workflow_rev](https://www.lexogen.com/wp-content/uploads/2017/02/06quantseq_workflow_rev.jpg)
During the library amplification step sequences required for cluster generation are introduced.
![07quantseq_workflow_rev 07quantseq_workflow_rev](https://www.lexogen.com/wp-content/uploads/2017/02/07quantseq_workflow_rev.jpg)
Multiplexing can be performed with up to 384 barcode combinations using the available 96 i7 indices and four i5 indices.
Step 5: Sequencing
![08quantseq_workflow_rev 08quantseq_workflow_rev](https://www.lexogen.com/wp-content/uploads/2017/02/08quantseq_workflow_rev.jpg)
NGS reads are started at the very last nucleotide of the mRNA using the Custom Sequencing Primer (CSP Version 2, included in the kit) for sequencing. The reads generated during Read 1 reflect the cDNA sequence. With QuantSeq REV also paired-end sequencing is possible.