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Old 11-25-2009, 03:41 AM   #1
aleidenroth
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Default Covaris S2: Chromatin shearing for ChIP(-seq?)

Hello everyone,

I'm new to this forum and have just started a PhD.

I'm planning to ChIP an epitope tagged protein (a transcription factor I'm expressing exogenously in transient transfections).

Currently, I'm testing shearing conditions on the Covaris S2 - has anyone used it to shear chromatin for ChIP applications? The lab who owns it here just uses it for Deep-SEQ library preps.

I can get the DNA into a size range of 200-700 quite happily, although the sample gets very warm during sonication. Here are the conditions that I use:

Sonication volume: 500ul

Treatment 1:
Duty Cycle: 20%
Intensity: 8
Cycles per burst: 200
Cycle time: 60 seconds

Treatment 2:
Rest (to prevent warming)
30 seconds

Cycles: 20

Tube gets warm even after only one Treatment 1 60 second exposure. Will this affect protein integrity or cross-linking, and if so, should I try and drop the sonication time to 30 or even 15 seconds? How about dropping the intensity?

Also, what size range is suitable for downstream ChIP-seq? Depending on where I look, suggested size ranges vary quite a bit.


Thanks in advance for any feedback / nuggets of wisdom.

Andy
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Old 11-25-2009, 06:26 AM   #2
ECO
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Check out the microtubes and the newest protocols for <1kb:

http://covarisinc.com/supported-protocols.html

20 minutes is pretty hard on your DNA, not to mention the damage to the crosslinks/proteins. You will be able to get the same size ranges with 1-3 minutes with 1/2 the intensity.

I'm sure Hamid will chime in soon.
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Old 11-25-2009, 09:42 AM   #3
Hamid
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Hi Andy,

I do have a few questions:
1. Which tubes are you using for the processing of your 0.5ml samples?
2. What is the cell number you are using in the 0.5ml sample?
3. Are you preparing the nuclei prior to shearing?
4. What is the constituents of the shearing buffer?
5. What is the protocol you are using for preparing your cells for ChIP?
6. Did you carryout a time course shearing to determine the optimal shearing time?

We do have a protocol for chromatin shearing. It is located at http://covarisinc.com/pdf/pn_400066.pdf. please take a look, and let me know if you have any other questions. Please feel free to contact me directly.

Thank you.

hamid
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Old 11-27-2009, 12:33 AM   #4
aleidenroth
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Thanks both.

@ECO
We have tried the <1.5kb conditions but got pretty big fragments.
I'll try and drop the intensity though. To get the time down, would you drop cycle time or cycle number?

@hamid
1) 1.5ml standard eppendorfs
2) 4x 10^6 cells, although I might increase that. Spending a lot of time waiting for my cells to grow at the moment...
3) I first lyse cells (5mM PIPES, pH8, 85mM KCl, 0.5% NP-40) and then nuclei (50mM Tris-Cl pH8, 10mM EDTA, 0.8% SDS)
4) I shear cells in the nuclear lysis buffer from 3)
5) Do you mean after the sonication? I haven't done any IPs yet, still at a very early stage.
6) We tested different cycle numbers (5, 10, 15, 20, 25, 30), and actually they all look pretty similar. We get slightly lower smears with higher cycle numbers.

Thanks for the linked protocol, I had seen that but used the other one (for SDS-buffers), which now seems to have disappeared off the website.

I'll try those conditions and see how low I can drop the cycle number.

Thanks for the quick feedback,

Andy
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Old 11-27-2009, 10:01 AM   #5
Hamid
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Hi Andy,

I have definitely identified the problem you are having.
Please realize that our sample vessels are an integral part of our acoustic technology. We have invested a significant energy and time in their development to ensure that they do not interfere in the delivery of controlled acoustic energy to your samples. The use of the polypropylene tubes are not recommended for any of our shearing protocols requiring high energy delivery. The reason for that is that polypropylene, and polystyrene absorb as well as deflect a significant amount of the acoustic energy during high energy processing. This translates to heating of the tube, which in turn heats your sample. The absorption and deflection also greatly reduce the energy delivery to your samples.
As suggested in the protocol, http://covarisinc.com/pdf/pn_400066.pdf, please use either the TC12, or TC13 tubes depending on your sample volume. These glass tubes are specifically designed and validated for efficient delivery of controlled acoustic energy to your samples.

I also wanted to note that the protocols we have optimized and validated for shearing of naked DNA, are not transferable or usable for the shearing of chromatin.

Thank you

hamid
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Old 12-04-2009, 12:27 AM   #6
aleidenroth
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Hi Hamid,

Thanks for the advice. I have switched to TC13 tubes. I know I should be using TC12 for 0.5ml volume, but at the moment we don't have a holder for those. Do you know if there's a glass tube product we could buy to use in the THQ-MT-0.65 or THQ-MT-1.5 holders?

Samples stay nice and cold in the 13x65mm tubes and the size range looks pretty good too - so thanks very much!

Andy
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Old 12-04-2009, 06:45 AM   #7
Hamid
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Hi Andy,

I am glad that it worked out for you. If possible, can you please email or post a gel image of your chromatin shearing results.

Thank you

Hamid
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Old 12-07-2009, 02:34 AM   #8
aleidenroth
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Default gel pic

Hi Hamid,

here's a gel pic. Conditions are as above except treatment 1 is 30 seconds sonication rather than 60s, and intensity is 5 rather than 8.

I think 25 cycles (12.5 minutes exposure) looks okay for a start.

Ladder (from bottom) is 200bp, 400, 600, 800, 1kb.

Sorry, the scan's not great.

Regards

Andy
Attached Images
File Type: jpg sonication jpg.jpg (61.4 KB, 365 views)
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Old 01-16-2017, 07:06 AM   #9
mohande
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Hi all,

I am trying to shear chromatin using Covaris S. I have tested 6 different conditions on Genomic DNA first and what it works for me was (20%, 10, 200, 15 min), but when I apply to chromatin it gives me a very large size range. I have used 12x24 tubes, not the 130 ul ones.

Here are 6 conditions I have applied to chromatin:
duty cycle 20%, 20%, 20%, 20%, 20%, 20%
intensity 10, 10,10,10,10,10
duty per bust 500,200, 200, 200, 200, 200
time (min) 30, 20,30,40,50

Can anybody tell me how can I shear my chromatin in the range on 200-700 bp.

Cheers :-)

ps: if that helps I am using this tubes:
http://covaris2.corecommerce.com/Con...m-100-p43.html

Last edited by mohande; 01-16-2017 at 07:56 AM.
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