regarding MboI enzyme of in-situ Hi-C

[feife...@gmail.com]

Hi,

We are trying to follow the in-situ Hi-C protocol as published in Cell, Rao et al.,2014. I have two questions about MboI digestion.

1) Why did you choose MboI as the restriction enzyme? According to my understanding, any 4-cutter with blunt end will work in the in-situ Hi-C protocol. 

2) Some of the MboI restrcition fragments of cross-linked nuclei are very long in our experiment. Could you show me a gel of MboI digestion and ligation after reversal?

Thank you very much for your help!

Feifei 

[Suhas Rao]

Hi Feifei,

I apologize for the delayed response!

re 1) actually restriction enzymes with 5' overhangs are desired, as the biotinylated nucleotide is incorporated via a fill-in step. MboI was chosen because we saw the best preliminary results with it in a screen of various 4-cutter enzymes while developing the in situ Hi-C protocol. However, since then we've used a variety of different 4-cutter restriction enzymes, for instance DpnII and MseI also work well.

re 2) in our experience, it is not possible to get complete digestion on fixed chromatin and some long uncut fragments will remain after digestion (even after 24 hours with excess enzyme). However, you should see a fairly bright smear of digested DNA <1kb in length after digestion.

Hope that helps,
Suhas

[Fei Lu]

Hi Suhas,

I performed MboI digestion following your protocol, but only got big (>1kb) fragments. I am wondering if you could provide some more details on the MboI digestion products or if you mind sharing a gel picture.

When you said "should see a fairly bright smear of digested DNA <1kb in length after digestion",  around what size range of a smear should I be expecting? And also, how much gDNA would you estimate to be digested to be <1kb, 10%, 30%?

Thanks for your time and help!

Best,

Fei

[Elena Stamenova]

Hi Fei,

We do not normally run gels on the restricted material as sufficient portion of the nuclei have to be lysed, cross-links completely reversed and material purified in a way that does not create any size bias. 

We find it hard to draw conclusion about the success of a HiC experiment based on such gels since crosslinked chromatin is not fully accessible and is normal for longer fragments to present after restriction. We have not attempted to quantify % of DNA in certain size ranges after restriction.

I would suggest  that you finish your HiC library and sequence 100K-1M reads from it and look at number of metrics in order to assess library quality as detailed in section II.d of the Extended Experimental Procedures of Rao & Huntley et al, Cell 2014.

Best regards,

Elena

[Fei Lu]

Hi Elena,

Thank you very much for your quick response. Really appreciate it!

Cheers,

Fei

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