How is strandedness preserved with PCR (DNA/RNA-seq)

Question

[Sotiris]

I understand that during the library preparation by the dUTP method, the Us on the second cDNA strand "mark" it in a way that is then depleted, so then only the first cDNA strand is amplified via PCR. I don't understand how that method allows us to retain the information of the originating strand of the fragment. Isn't PCR just amplifing the remaining cDNA strand by synthesis, therefore creating complementaries? And then the resulting library will again have half cDNAs that correspond to one strand and half that are reverse? How then can we call the strand of the original fragment?

Answer 1

[Carlo Yague]

Briefly, directionality is preserved because of the dUTP method and because the 5' and 3' adapters that are ligated to the cDNA have different sequences.

Let me explain: because of the dUTP method, only the first cDNA strand [5'-5'adapter-insert-3'adapter-3'] is amplified by PCR. After amplification from the adapters, we also have the reverse complement (RC) of that: [5'-RC(3'adapter)-RC(insert)-RC(5'adapter)], but as you can see, the 5' and 3' ends are different. Therefore, it is easy to discriminate the strands when the sequencing starts.

In contrast, if you do no not do the dUTP method, then you amplify both [5'-5'adapter-insert-3'adapter-3'] and [5'-5'adapter-RC(insert)-3'adapter-3'] and lose strand information immediatly.

The difference is well illustrated on this blog post: azenta.com/blog/stranded-versus-non-stranded-rna-seq