No peaks

[chipmonk]

I am getting good alignment to the genome after illumina chipseq but when i call for peaks there are no significant peaks. and the FDR is like 100% for all, even the IgG control. Can i narrow down to whether the antibody or the conditions for binding are not good based on chipseq data!

I donot have a method to verify the pulldown as there are no targets. any suggestions for this, chip western also doesnt work.

Is it ok to crosslink the nuclei prep as the tissue seems to have issues with penetrance?

please do suggest..thanks

[mudshark]

too many things that could have gone wrong. if there are no known binding sites of your target, ChIPSeq from tissues is maybe not a very good idea. why not establish the technology in a cell line before moving to tissues?
is the tissue freshly prepared or frozen?

[chipmonk]

Thanks for the reply. The tissue is fresh, dissected into PBS with Protease inhibitors and then as the tissue penetrance seems less I isolate nuclei and fix. The antibody is specific and polyclonal which shows good titre at least on western. The experiment is to understand the binding of the TF and its role in development hence cell line may not be a great idea. any other suggestions...

[mudshark]

I guess your problems center around chromatin preparation and ChIP. Is there anything known about binding sites of your TF in any other tissue? I think you should should set up your ChIP from scratch and if this works move on to tissues and then do ChIPSeq.

[chipmonk]

Thanks mudshark for the suggestion. I am already on that track but there is nothing known about the tf s targets in the organism. i could try the same protein in other models and get back! or a positive control like histone may be useful. I just was looking at the points where I could troubleshoot!