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ChIP- Unwanted signal from intergenic region

ChIP Intergenic negative control

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#1 belew

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Posted 08 October 2013 - 01:17 AM

Hi,

 

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.



#2 Mighty Mouse

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Posted 18 November 2013 - 03:03 PM

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.

 

MM


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