Can local chromatin structure affect sonication? - (Apr/11/2013 )

Hi all,

I've been looking at various regions of the interleukin-2 promoter in Jurkat T cells using ChIP. I recently got some data that doesn't make much sense to me, and I'll try to convey it the best I can. So we're looking at two different regions of the IL-2 promoter and I got data that looks like this:

Region 1 Input Cts
Stimulated Sample 1: 25
Stimulated Sample 2: 25
Unstimulated Sample 1: 25
Unstimulated Sample 2: 25

Region 2 Input Cts:

Stimulated Sample 1: 25
Stimulated Sample 2: 23
Unstimulated Sample 1: 25
Unstimulated Sample 2: 25

Each sample had the same number of cells go into the IP, and therefore in the input, so it makes sense that the Cts for input are basically the same for all the samples. The obvious question is how could I get a different ratio of input Cts between two regions in Stimulated Sample 2 (25 vs. 23, where all others are 25 vs. 25). To me it almost suggests that my amplicon in Stimulated Sample 2 is more protected during the sonication step and therefore more of it survives and is able to be amplified. Is there any precedence for this type of thing being an issue, or has anyone encountered anything similar in their own work? Thanks for any suggestions

Hi Evan,

Are sample 1 and sample 2 biological replicates, or they represent a different condition?

Vassil on Thu Apr 11 20:30:23 2013 said:

They represent different experimental conditions (Sample 1 was treated with a scrambled siRNA, Sample 2 was treated with a "real" siRNA).

Hi Evan,

This is a strange situation...

Since you are just taking the input, then I don't think it matters how protected the chromatin (or DNA fragment) is. The sonication does not destroy the DNA (correct me if I'm wrong). All this to say that the input values should be the same.

It seems to be that the problem arises from the qPCR reaction itself somehow.

Please keep us posted about any solutions (even if it is just messing up with the loading of the sample).


Good luck!