Hi everybody,
I am planning to perform ChIP experiments and sequencing on fibroblasts that reside in a 3D collagen/matrigel gel. Does anyone have experience with fixing and subsequent harvesting of cells and/or nuclei from 3D gels and the isolation of chromatin.
My cells will be seeded at high concentration in the gels for a couple of days and after that we will perform ChIP experiments for a transcription factor.
Some specific questions:
- Does formaldehyde pose any difficulties with cross-linking of cells to the ECM?
- Is the collagen/matrigel easy to remove? Do I need to use proteases to get rid of it?
Any help is much appreciated!
Thanks
Hi Bram,
I can't answer all your questions but have some tips for some of them.
To fix cells in matrigel you should follow the protocol of any supplier. According to one I read, Formaldehyde should diffuse easily into the gel. Nuclei isolation should not matter if there is still residual ECM in your initial sample.
I would imagine you want to use a low cell number based ChIP procedure. Take a look at either of the 2 following papers/protocols:
1) From the Henikoff lab https://www.nature.com/articles/nprot.2018.015
this uses unfixed cells, so when you are able to obtain your cells fast and cold from your gels this could be a great method.
2) When fixation is a must use the following: https://www.nature.com/articles/s42003-018-0219-z
The author use enzymatic digestion of chromatin instead of sonication which should preserve >60% of your TF epitopes so it will increase the yield after ChIP.
Hope it helps you out ;)
An alternative to the above protocols could be the following kit from Active Motif:
https://www.activemotif.com/catalog/1218/tam-chip
But instead of using fixed cells (as the protocol mentions) use unfixed cells so you dont have to sonicate and let the Tn5 transposes (from the kit) fragment the chromatin. This way you will end up in a lot higher pull down efficiency. You could start with 100.000 - 1x10^6 cells per antibody.
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