Hello,

You may consider using less cycles or less power per cycle for non-crosslinked samples because native chromatin is usually sonicated more easily than the crosslinked one.

Then, What could be the reason that I have less DNA? Is the DNA broken in very small peaces that I can not detect in a gel?

Thanks in advance Anatoliy

What do I think:

1. Some non-specific staining (e.g. not only the DNA is stained with EtBr after crosslinking). Or you may have the RNA from ribonucleoproteins being crosslinked and stained alondside with DNA. In this case you may just see more stain in protocol with crosslinking.

2. The DNA is lost from nucleosomes more easliy and you obtain less material after sonication as DNA is fragmented into smaller pieces.

3. In crosslinking you may inactivate nucleases which will make the degradation of DNA slower.

To quantify DNA independently of staining, you may try running qPCR on i) crosslinked-sonicated-decrosslinked and purified DNA or ii) not crosslinked-sonicated and purified the same way. Ideally, use the same cells split in half.

I do not think is RNA what I see because in both casses I treat the samples with RNase A for 2h to eliminate most of the RNA from the samples. But the inactivation of the nucleases in crosslinked samples and that maybe in not crosslinked samples the DNA is beeing broken into smaller pieces that I do not see in the gel are good points. I will quantify by qPCR to see wich is the real difference between the samples and I will look for sonication protocols especific for not-cosslinked samples.

Thanks for your answer!

Mar Bustamante Sequeiros you may try adding some artificial DNA (e.g. purified PCR amplicon from qPCR reaction) to your crosslinked sample, non-crosslinked sample and handling control sample (containing no cells, but processed the same way) to see whether your PCR amplicon is preserved throughout sonication to the same extent. Drawback: using PCR amplicons together with sonicator is really messy thing ))