Step 1: Reverse Transcription

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The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.

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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

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After first strand synthesis the RNA is removed.

Step 3: Second Strand Synthesis

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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.

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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

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During the library amplification step the sequences required for colony formation are introduced.