What does the final RNA-Seq library for sequencing by Illumina contain? ss cDNA or ds cDNA?

Question

[MCH]

• 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?

Answer 1

[ATpoint]

The library itself is double stranded as standard PCR always produces dsDNA. The library is denaturated using NaOH (at least on the MiSeq, on the larger machines I never did it myself, probably the same process) prior to loading onto the flow cell resulting in single-stranded DNA.

[MCH]

Thank you for your answer. Then, why isn't included in the TruSeq protocol? Even when I read scRNA-seq protocol ( like Mars-Seq 2), the last step in library prep is again elongation.

[ATpoint]

Because this is part of the sequencing protocol, not of the library prep. Don't do that yourself! The sequencing facility will do that when you submit the sample. THe last step of a PCR is always terminal elongation to ensure all molecules are fully elongated. When you read e.g. the MiSeq sequencing protocol you will see that there is a denaturation step.

[MCH]

Thanks for your answer :-) you are right. I just read NextSeq 500 System Guide. The denaturation step is done by lab technician.