Step 1: Library Generation
The starting material is either rRNA depleted RNA or Poly(A) selected RNA.
Library generation starts with the random hybridization of the starter/stopper heterodimer mix to the poly(A) selected or rRNA depleted RNA.
A single-tube reverse transcription and ligation reaction extends the starter to the next hybridized heterodimer, where the newly synthesized cDNA insert is ligated to the stopper.
RNA or cDNA fragmentation is not required as the insert size is dictated by the distance between starter/stopper binding sites.
During second strand synthesis the RNA is hydrolyzed and the library is converted to double-stranded DNA. The double-stranded library is purified using magnetic beads to adjust the library length and get rid of second strand synthesis reaction components.
Step 2: Library Amplification
The library amplification is performed to add the complete adaptor sequences required for cluster generation and to produce sufficient material for quality control and sequencing. i7 and i5 indices can be added during this step in order to be able to uniquely multiplex your samples for the sequencing run.
The finished library is purified from PCR components which can interfere with quantification.