Hi Marcela

We have used a protocol from this paper pretty successfully on cerebellar tissue. Unfortunately it can take a lot of trouble shooting to get the sonication cycles and time right, depending on your tissue.

Hope this helps

Cheers, James.

Article Chromatin Immunoprecipitation in Early Xenopus Laevis Embryos

Hi Marcela,

indeed IP from tissue is very tricky and difficult... What I could suggest, although I imagine you are doing it, is to make sure you start with a good homogenization of the brain tissue. I would homogenize in a classical buffer for synaptosomal preparation to remove all peripheral tissue, for example:

0.32 M sucrose

1 mM EDTA

1 mg/ml BSA

5 mM HEPES pH 7.4

Use large volumes, it will homogenize better (10-12 ml for a cortical hemisphere, 3 ml for striata or hippocampus, etc), and run it in a douncer at 600 rpm, at least 10 strokes, in ice, until you see a uniform milky solution. This will not lyse the cells, but will produce a sort of single cell suspension removing all extracellular matrix and dendritic and axonal projections. Then spin 10 min at 3000 g at 4 °C and discard the super. The pellet will be essentially the neuronal and glial somas. This you can lyse as if you were starting from a cell culture pellet that you say is working in your hands...

I hope it helps. Good luck.

I would highly recommend using enzymatic fragmentation as it is effective and much easier.

If you intend to use Tn5/nextera for library preparation, then chromatin fragment size is not really a concern and having somewhat larger fragments can improve sensitivity, especially if you are using low cell number as input.

take a look at https://academic.oup.com/nar/article/45/17/e153/4037354

Enzyme coktails or MNase give good results and are much easier to reproducue and optimize than sonication.