If you use random primers to make your first strand cDNAs, then it should not matter if teh RNA has been polyadenylated firsst. Although, I am not sure why you would want to do this.

Thank you all for your great answers. My interest is in vitro for technical not biological reasons. So far I tried ribozero after polyadenylation but the RNA quality was bad. I decided now to use ribozero first (though i have to use larger amount of RNA) then poly A.

Polyadenylation of RNA is used by some library prep methods help selection of fragments later, so adding them is not an issue. Unless you have labelled your RNA with something, what I see from your trace however is probably high molecular weight DNA contamination. Did you treat your total RNA extraction with DNAse ?

Thank you all for your answers. I think polyadenylation affects the ribosomal RNA depletion by RiboZero ( the picture above). Thus, I had to deplete the RNA first then polyadenylate it. Since I am using Truseq stranded mRNA kit, I used the poly T beads and the recovery was almost the same as the normal way (without polyadenylation).

Hi Mohamad Al Kadi ,

Did you ever successfully polyadenylate the bacterial RNA?

Or did you find that the polyadenylation did not work?

Morgan Sobol Yes, it worked fine. I used it to sequence bacterial RNA using Nanopore Direct RNA sequencing and Illumina (for comparison). Other authors also used it successfully.

Mohamad Al Kadi Thanks for the reply! That is good to hear and I am actually interested in polyadenylation for the Nanopore as well. Has your research been published yet? If so, I would like to read it to learn more :)

Morgan Sobol Not yet. I will let you know as soon as I published it.

Morgan Sobol Sorry for the late response. This is my paper:

https://journals.asm.org/doi/epub/10.1128/mSystems.00996-21