Question: Low input ChIP-seq on frozen tissue
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gravatar for evakoole
3 months ago by
evakoole0
evakoole0 wrote:

Dear all,

Currently I am working on a project to find the most optimal protocol for doing ChIP-seq on frozen tissues. Most protocols are designed for cell lines, however, ChIP-seq in vivo (small biopsies for example) is really interesting to me. Especially differences in transcription factors and histone modifications between various tissues are of great value for functional annotation of genes.

I have found a promising protocol with very low-input that targets RNA Pol II. My question to you is whether the input is always lower when you target RNA polymerase. I have no experience doing ChIP targeting anything else than TF and histone modifications.

Also, if anyone of you works with a certain protocol that works well, is easy, cheap, or has low input, could you please share this with me?

Thank you so much in advance!

Kind regards, Eva

chip-seq • 204 views
ADD COMMENTlink written 3 months ago by evakoole0

I would be most concerned about the protein quality. Most ChIP protocols have fixation as the first step to preserve protein structure and DNA interactions. Would antibodies continue to bind proteins from a thawed sample?

ADD REPLYlink written 3 months ago by jotan1.2k

Hi Jotan, thank you for your reply! For me, the biggest concern is keeping the nuclei intact until after the fixation step. To create better access for the formaldehyde, the tissue needs to be disaggregated (dounce homogeniser for example) If the nuclei are damaged, the DNA:protein interactions can change, resulting in faulty outcome of the experiment. The antibodies will for sure bind to the proteins of interest of a thawed sample (tried and it worked), sometimes adding more antibodies is necessary though. This will of course make it quite a bit more expensive. Why would you think the antibodies cannot bind to the proteins? Due to altered structure? I'll look into that and might try fixating the tissue before freezing, just to see what it does. Thanks!

ADD REPLYlink written 3 months ago by evakoole0
1

"Why would you think the antibodies cannot bind to the proteins?"

I was mostly thinking protein degradation.

I suppose the only way to find out is to try. Good luck!

ADD REPLYlink written 3 months ago by jotan1.2k

Okay thank you! When I find out more, I'll put it under this message but it will take a while before my experiments are done.

ADD REPLYlink written 3 months ago by evakoole0
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