Dear Robert,

yes, in my experience, RNA can obscure your DNA. In your case, it looks like you have both on the gel. The upper bands are your DNA and the lower ones are the RNA. Don't you do an RNA digestion for the ChIP fragmentation check? I'm not really familiar with the type of sonicator you are using, do you have any idea how your sonication relates to tip sonication and/or Bioruptor (Diagenode)? And of course, as you probably know, also the fixation time/temperature/concentration of formaldehyde affect your crosslinking and thus your sonication efficiency.

Best,

Kevin

I agree with Kevin, that it might be RNA. A simple RNAse treatment before proteinase K digestion should get rid of it. Another possibility is that if you are using loading buffer that has different dyes in it (such as Bromophenol blue) it might render part of the smear darker. For this reason I use pure 10% glycerol as a loading buffer when checking sonication efficiency on agarose gels.

I think I have found my problem. I was running the sonicated material on the gel before reversing the crosslinks. I performed an experiment on my sonicated sample where I reversed the crosslinks, then treated with RNase and then subsequently treated with proteinase K, or  just reversed the crosslinks and only treated with proteinase K. There was no difference between the + or - RNase treated samples, and the upper band from my posted image was not there, only the bottom band. I assume that the duration of heating in the water bath during the reversing of crosslinks was sufficient to destroy the RNA in all my samples, regardless of RNase treatment or not. It turns out our sonication seems appropriate for ChIP. Thanks for your helpful suggestions.