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 02quantseq_workflow](https://www.lexogen.com/wp-content/uploads/2015/04/02quantseq_workflow.jpg)
Library generation starts with oligodT priming containing the Ion Torrent-specific P1 adapter in the 5`part of the oligodT primer.
Step 2: Removal of RNA
![03quantseq_workflow 03quantseq_workflow](https://www.lexogen.com/wp-content/uploads/2015/04/03quantseq_workflow.jpg)
After first strand synthesis the RNA is removed.
Step 3: Second Strand Synthesis
![04quantseq_workflow_it 04quantseq_workflow_it](https://www.lexogen.com/wp-content/uploads/2017/02/04quantseq_workflow_it.jpg)
Second strand synthesis is initiated by random priming and a DNA polymerase. The random primer contains the Ion Torrent-specific A adapter in the 5’ end of the random primer and up to 48 in-line barcodes can be introduced.
![05quantseq_workflow_it 05quantseq_workflow_it](https://www.lexogen.com/wp-content/uploads/2017/02/05quantseq_workflow_it.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_it 06quantseq_workflow_it](https://www.lexogen.com/wp-content/uploads/2017/02/06quantseq_workflow_it.jpg)
During the library amplification step the sequences required for colony formation are introduced.