Step 1: Reverse Transcription
The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.
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
After first strand synthesis the RNA is removed.
Step 3: Second-Strand Synthesis
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.
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
During the library amplification step sequences required for cluster generation are introduced.
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).