Step 1: Reverse Transcription
![01quantseqflex_workflow 01quantseqflex_workflow](https://www.lexogen.com/wp-content/uploads/2015/06/01quantseqflex_workflow.jpg)
The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.
![02quantseqflex_workflow 02quantseqflex_workflow](https://www.lexogen.com/wp-content/uploads/2015/06/02quantseqflex_workflow.jpg)
Library generation starts either with oligodT (included in the kit) or a target-specific primer containing sequencing platform – compatible linker sequences.
Step 2: Removal of RNA
![03quantseqflex_workflow 03quantseqflex_workflow](https://www.lexogen.com/wp-content/uploads/2015/06/03quantseqflex_workflow.jpg)
After first strand synthesis the RNA is removed.
Step 3: Second-Strand Synthesis
![04quantseqflex_workflow 04quantseqflex_workflow](https://www.lexogen.com/wp-content/uploads/2015/06/04quantseqflex_workflow.jpg)
Second strand synthesis is initiated by random (included in the kit) or target-specific priming and a DNA polymerase. The primers also contain sequencing platform – compatible linker sequences.
![05quantseqflex_workflow 05quantseqflex_workflow](https://www.lexogen.com/wp-content/uploads/2015/06/05quantseqflex_workflow.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_upd 06quantseq_workflow_upd](https://www.lexogen.com/wp-content/uploads/2017/02/06quantseq_workflow_upd.jpg)
During the library amplification step sequences required for cluster generation are introduced.
![07quantseq_workflow_upd 07quantseq_workflow_upd](https://www.lexogen.com/wp-content/uploads/2017/02/07quantseq_workflow_upd.jpg)
Multiplexing can be performed with up to 384 barcode combinations using the up to 96 available i7 indices included in the kit and four additionally available i5 indices from the i5 Dual Indexing Add-on Kit (Cat. No. 047).