Prominent non-rRNA Bioanalyzer peak after rRNA depletion

[arctan]

Has anyone experienced seeing a prominent non-rRNA peak after rRNA-depletion? I am seeing this consistently in my samples.

See the attached image for an example, which shows Bioanalyzer RNA pico before (top) and after (bottom) rRNA depletion by RiboZero.

These are bacterial RNA samples, so the main peaks in the total RNA are 23S and 16S (not 28S and 18S as annotated by the program). After rRNA depletion, the rRNA peaks are pretty much gone, but there is a very sharp, prominent peak running at about 400 nt.

Anyone have ideas on what this could be? It is just a very strongly expressed housekeeping gene? Or it it possibly something else?

[JakobHedegaard]

Hi Arctan,
We have also done some bacterial RNA-Seq using Ribo-Zero (and ScriptSeq) and have observed the same peak. I have "only" generated the data, but have not received any feedback from the "customer" regarding a dominating transcript following their analysis.
I suggest that you go ahead and sequence ;-)
Best,
/Jakob

[arctan]

Thanks for replying!

I am not sure what the peak is, but it seems reproducible because, I've seen it in all of the different RNA preps I've done where I also ran Bioanalyzer after rRNA-depletion.

The sample I showed was a moderate case. For some samples, the peak is there but less prominent. For other samples, the peak is even stronger.