In-Situ Hi-C Protocol

[dozt...@gmail.com]

Hello everybody,

I have a question about the in-situ Hi-C protocol, in general.

After the cell permeabilisation step, (before the digestion) is it OK to treat the sample with first 10% SDS and then Triton X-100 and then continue with digestion? Or should I directly continue with the digestion? I found this protocol and it says to use SDS and Triton and go the digestion from there. 

But when we use SDS and Triton, we would open the nucleus and it won’t be in situ anymore, right? This where I’m lost, actually.

Thanks!

Best Regards,

Dogancan

[Elena Stamenova]

Dear Dogancan,

We use 0.5%SDS to permeabilize nuclei before digestion (same protocol you linked in your question). Nuclei remain intact after this step and SDS is quenched with Triton-X100 before addition of restriction enzyme.  I definitely  would not recommend using  higher SDS concentrations as this will compromise nuclear integrity. 10% SDS is very high concentration and should not be used for in situ Hi-C preparations.

Best regards,

Elena

[dozt...@gmail.com]

Thank you very much!

Dogancan

24 Mayıs 2018 Perşembe 06:09:29 UTC-7 tarihinde Elena Stamenova yazdı: