Did I do this right? Testing efficiency of poly(A) tailing.

Question

[Morgan S.]

Hi there,

I am trying to determine how efficient my poly(A) tailing is. I am using NEB's E.coli Poly(A) polymerase to further add poly(A) tails to RNA from E.coli. I sequenced Total RNA from a sample with and without polyadenylation to compare the efficiency of the tailing. After an initial trimming with TrimGalore to remove adapters and bad quality bp, I did another trimming on the trimmed paired reads where I designated the adaptor sequence as -a {A}10 (10 A's). If this was the right thing to do, then my polyA tailing did not work since both samples had 16% of the reads containing A tails with at least 10 A's. I would just like to know if I did the right thing to find polyA tails in my sequences.

Thanks! Morgan