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Sonication Vs MNase - (Apr/14/2011 )

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I am sure that this debate has been covered before, but I just wanted to share my own personal experiences with it. I am working with a TF and fairly recently switched to MNase. I initially tried MNase digestion out of pure curiousity, but I have to say that I (and my experiments) have not looked back since. I was deeply unhappy with sonication as it just didn't feel right and my experiments were not reproducible to the level that I would have liked.

I have found that MNase fragmentation is superior to sonication in EVERY way. I am talking better %input, better specificity, lower background and much, much, much higher reproducibility. I have even compared the two in ChIP-Seq experiments and there is a night-and-day difference between the two (tighter peaks, much less noise/bias). The only downside is that a lot of optimization is required but, once you are over the initial hurdle, it is so much easier in my hands.

I am sure that most of you will respond with the usual "yeh but you should use sonication for TF, but MNase for histones etc". But I think this dogma is just that, dogma. If you get the MNase shearing right (i.e. not down to single nucleosomes), you will be amazed at the results. Seriously, try it!

Just wanted to see how many others are starting to use MNase for TF ChIP?

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-Dukey-

Dukey,

I am glad to hear that someone is really willing to vouch for MNase. I really want to switch to it before i start ChIP-chip or ChIP-seq, whichever one I can justify doing. Can you elaborate on the optimization step and explain why it is important not to have single nucleosomes? Thanks for the post.........personally, the variability of sonication makes me sick.

-chabraha-

Sure!

Once you get down to single nucleosomes (<150 bp), you really start to see dramatic drops in % input values. I think that this is mostly because you are working blind in the primer design stage (at least I was) and so you may not be right on top of the binding site and you really have no flanking region to "buffer" the PCR. So your chances of capturing the binding event dimish, unless of course your primers are right on top of the site. If you go for slightly larger range of fragments (i.e. one to six nucleosomes) you see a huge jump in % input and I think it is related to the primer design point above. Note that there is really no parallel increase in background with less fragmentation.

I am convinced that this over-digestion problem is why MNase digestion has gotten a bad rap with TF ChIP. Most of the objections to it are also theoretical in nature and there is really little data to suggest it is inferior to sonication. In fact, all of my data suggests the complete opposite, as does a whole bunch of data from some vendors who are pushing it (i.e. Cell Signaling Technology). Once optimized, it is also very reproducible, assuming experimental conditions are kept consistent.

In terms of optimization, it is quite simple. You just have to titrate very carefully the MNase and be very careful to keep cell number consistent. In my hands MNase was very potent in chopping up my DNA and I ended up using a very small quantity in my reactions. One big simple optimization experiment should take care of it. I would be more than happy to give you some more details should you decide to switch.

I am submitting my ChIP-Seq data for publication this week and so we will see how it copes in peer review. I am not anticipating any major issues with the ChIP methodology.

-Dukey-

Good to know about the vendors pushing it....I will buy from one like cell signaling I'm sure. Right now I am waiting on a cell line and have to do a few tests on it before I can do my ChIPs, but if everything goes well I will for sure be contacting you. Best of luck on the manuscript!

-chabraha-

Hey Dukey,

I was wondering which kit you are using for the MNase. We are currently trying the SimpleChIP kit (Cell Signaling), but we are having problems with the sonication step (too bad it still needs sonication).

How many cells do you normally use, how much MNase, how much lysis buffer, which type of sonicator?

Hope you can help, because we are really struggling right now.

Cheers,
Roel

-roelq-

OK, so here is my brief protocol. I use the SimpleChIP enzymatic magnetic kit from CST (#9003).

- Harvest 5 x 10(7) cells per experiment. For me that is about 4 x 10cm dishes of cells.
- Follow protocol exactly (except for 30 min protein lysis on ice), up until the addition of MNase
- I add just 0.3 ul of MNase/5 x 10(7) cells in 1 ml of buffer B and leave for 20 min at 37 degrees
- The protocol states that 5 ul of MNase is what CST used for HeLa cells. When I tried this amount, I had nothing but single nucleosomes.
- I then resuspend the nuclear pellet in 1 ml ChIP buffer as per the protocol and split into two 500 ul aliquots
- I then sonicate very mildy just to break open the nuclei. For this I use a Branson Sonifier on 20% and do 2 x 10 second pulses for each aliquot.
- 100 ul of the clarified chromatin is then used in each IP reaction (final volume 500 ul). Therefore, I use about 5 x 10(6) cells per IP reaction. This is pretty much per the protocol
- IPs are left O/N and then I follow the protocol exactly from there on in.
- For ChIP-Seq, I will usually do 4 - 5 individual ChIPs for my TF and then combine at the DNA purification step to get enough material for quantification and library prep.

Hope this helps a little.

-Dukey-

Interesting post. I always shied away from the MNase because I heard from others who tried it that it simply resulted in too small of fragments, as you suggested. I wonder if it would work as consistently in tissue samples in which it is much more difficult to determine and control the number of cells harvested and thus to obtain consistent optimization.

Interesting discussion.

MM

-Mighty Mouse-

Thanks a lot.

I am wondering if you ever tried to use lower cell number. We are currently trying with 4 x 10^5 cells because there is no way we would ever get 10^7 cells for our purpose.

One of our problems is that we seem to lose our DNA during column purification. So now that you say that you use only like 0.3 Ál MNase for 120x the amount of cells that we have, I guess we are just overdigesting our DNA and it is just too small to stick to the columns. Would you agree with that?

Cheers,
Roel

-roelq-

I have not tried lower cell numbers but I guess it could work, as long as you titrate the MNase very carefully. You may need to dilute it to make it easier.

-Dukey-

Dukey,

In a not so related question......What about MNase digestion on viral chromatin, which has a low nucleosomal content compared to cellular chromatin? Any down (or up)-side to using MNase on irregularly chromatinized with low nucleosome content?

-chabraha-
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