Step 1: Poly(A) Selection
During the poly(A) selection step the total RNA samples are briefly heated to resolve secondary structures and promote efficient hybridization.
The denatured total RNA is incubated with the oligodT beads, which specifically bind polyadenylated RNAs. RNAs lacking a poly(A) tail are then washed away, leaving only purified poly(A) RNA hybridized to the beads.
Step 2: Library Generation
As a result of this highly specific bead-based poly(A) selection step almost all traces of cytoplasmic rRNA, tRNA, and other non-polyadenylated RNAs are removed.
Library generation is based on Lexogen’s proprietary strand displacement stop/ligation technology. The starter/stopper heterodimer mix is randomly hybridized to the poly(A) RNA still bound to the magnetic beads.
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.
Distance is modulated by two different buffers resulting in different conditions for the reverse transcription/ligation reaction
During second strand synthesis the RNA is hydrolyzed and the library is converted to double stranded DNA. The double-stranded library is purified to remove magnetic beads and second strand synthesis reaction components.
Step 3: 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.