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Sonication Vs MNase


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#1 Dukey

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Posted 14 April 2011 - 09:59 AM

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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#2 chabraha

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Posted 15 April 2011 - 06:30 AM

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.
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#3 Dukey

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Posted 18 April 2011 - 11:12 AM

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.

#4 chabraha

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Posted 18 April 2011 - 06:57 PM

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!
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#5 roelq

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Posted 19 April 2011 - 01:21 AM

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

#6 Dukey

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Posted 20 April 2011 - 08:20 AM

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.

#7 Mighty Mouse

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Posted 20 April 2011 - 02:47 PM

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.

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#8 roelq

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Posted 21 April 2011 - 04:31 AM

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

#9 Dukey

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Posted 12 May 2011 - 11:44 AM

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.

#10 chabraha

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Posted 13 May 2011 - 01:17 PM

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?
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#11 nanook

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Posted 06 July 2011 - 07:47 AM

my major concern regarding MNase-ChIP is the chromatin release. are you sure you get a representative fraction of chromatin released from the nucleus? did you sequence your input chromatin, how evenly do the reads distribute?

according to my expts a huge part of chromatin remains in the nuclei after digestion with the released one being mainly chopped down to mono/di-nucleosomes.

i would be really interested in trying the MNase approach but without a proper release this technique is useless.

#12 Dukey

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Posted 03 August 2011 - 12:01 PM

my major concern regarding MNase-ChIP is the chromatin release. are you sure you get a representative fraction of chromatin released from the nucleus? did you sequence your input chromatin, how evenly do the reads distribute?

according to my expts a huge part of chromatin remains in the nuclei after digestion with the released one being mainly chopped down to mono/di-nucleosomes.

i would be really interested in trying the MNase approach but without a proper release this technique is useless.


Useless????? Wow.

The answer to your question is YES I did sequence the input chromatin and the reads distribute pretty nicely. In fact, the results looked a whole lot better than with sonication. That is for certain. Actually when you compare my data to other published studies, my results are VERY consistent. Actually the paper is in press now so obviously the reviewers didn't have a problem with my technique.

As I stated in my original post, the reason that MNase has gotten less attention is because of people like you following the crowd. If you digest down to mono-nucleosomes, then that is what you will get. However, if you do the proper experiments before hand and ensure that this doesn't happen, I personally believe that you get a good chromatin prep - or "proper release" as you call it.

How do you know you get "proper release" when you blast the crap out of your DNA-protein complexes with a sonicator? How do you know you don't destroy epitopes with sonication?

#13 wangjing

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Posted 03 August 2011 - 12:28 PM

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.


hi, What MNase are you using? Cat. No? and what buffer condition? I really want to switch to MNase soon! thanks!

#14 Dukey

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Posted 04 August 2011 - 10:06 AM

I am using the SimpleChIP Enzymatic Chromatin IP Kit #9003 (Cell Signaling Tech). Everything you need is in there and, apart from the modifications I have already mentioned, I pretty much follow the protocol.

Edited by Dukey, 04 August 2011 - 10:06 AM.


#15 angelawu

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Posted 22 August 2011 - 05:54 PM

Hi Dukey (and others),

Thanks for this post. I've been working with MNase for years now, but not been able to optimize it well enough for a good prep - caveat though, is that I'm trying to do it for thousands of cells instead of millions of cells, in which case the enzyme kinetics might be a lot different. My question for you is, do you know what buffer is used for lysing the cell prior to MNase treatment? The protocol from cell signalling doesn't give the specific buffer conditions. I'm just curious as to what buffer would be compatible with both sufficient cell lysis to release the chromatin for digestion, but also allow the enzyme to still function... For me, I think my buffer does the job for both, but badly, which is why my chromatin prep isn't so great.

Theoretically speaking, overdigestion with MNase of the chromatin into mono-nucleosomal fragments would give you bad qPCR results because you may not capture the protein binding site with your primer since the fragment length is smaller than your amplicon. However, this should be a non-issue if you are sequencing. So my question is, if you are just sequencing your chromatin-IP and not doing qPCR, does it even matter if the digestion has gone to completion and all the chromatin is in mono-nucleosomal fragments? Surely, for histone analysis this is even beneficial because it would give a higher resolution of the histone binding site? Anyone care to comment on this, or maybe have any experience with it?

Thanks in advance.

Edited by angelawu, 22 August 2011 - 06:00 PM.





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