I guess the oligos on Illumina's flow cell only hybridize to to single-stranded cDNAs (sscDNA).

However, when I read the TruSeq protocol (googled: TruSeq® RNASample Preparation v2 Guide) at the PCR step (AmpPCR, page 41) it looks like that the last cycle (d) is just elongation.

Therefore, since no denaturation (separation of dscDNA strands) happens, the final library input to Illumina should contain only dscDNAs which do not hybridize to flow cell's oligos!

My question is that then: when does denaturation happen? Inside Illumina? Or am I missing something?


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