ChIP- Unwanted signal from intergenic region - (Oct/08/2013 )
I have been doing ChIP experiments on brain tissues as well as immortalized cell lines (I use Diagenode's auto Chip kit). I see good enrichment for histone modifications and Pol II at promoters of my interest and very little background signal from my IgG control. However, I also see a substantial amount of signal from a desert intergenic region as well as other gene bodies (devoid of histone marks or Pol II binding site). Additional sonication steps could not help much.
Did anyone have a similar experience? Could tiering down antibody level solve the issue?
thanks a lot for your help.
Hi there...what are you using for downstream analysis? end point PCR or qPCR?
I think using an intergenic region can be difficult for something like pol II or a histone modification because you could very well have binding of pol II to such a site and/or histone modifications there. Much of the genome appears to be transcribed. Using "gene bodies" (which I assume you mean the middle of a gene?) wouldn't be good either for pol II since it is actively transcribing and thus would show up in the middle of a gene; ditto hisotne modifications.
if you are doing end-point PCR you can try other intergenic regions. If you are doing qPCR you could consider a region that is actively transcriptionally repressed e.g, LINE1 or some other retrotransposable element.