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Old 12-18-2012, 01:01 AM   #1
nfourier
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Default Genomic DNA shearing with Covaris E220

I am doing a ChIP experiment and doing the genomic DNA (RAW cell line, mouse monocytes) shearing using the Covaris E220. I need my DNA fragments to be between 500-1000 bps, but 200-700 bps will be ok too.
I first started doing a time-curve using the Covaris truChIP protocol:
duty cycle: 2%
peak incident power: 105 watts
cycles per burst: 200
temp: 4-8
volume: 130 ul (with covaris microtubes).
time points: 4,8,12 min.

My buffer is from the FastChIP protocol (which has been used before in our lab, and with the same type of cells and was successful), which contains: 150mM NaCl, 50mM Tris-HCl (pH 7.5), 5 mM EDTA, NP-40 (0.5%), Triton X-100 (1%). this buffer is used for lysis and shearing.

I get fragments between 1000-3000 bps.
I am totally new to Covaris and don't know what to change first in order to get smaller fragments. Should I increase the time? Or will changing the duty cycle first be more effective (and to what %)?
I could use all the help I can get.
Attached is the gel image (from left to right: 4,8,12 min. Ladder- bold from up to down: 3000,1031,500 bps).

Thank you!
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File Type: jpg 18.11 covaris 4-8-12min ONLy.jpg (68.7 KB, 42 views)
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Old 12-18-2012, 02:41 AM   #2
lorendarith
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Take a look at this:
http://covarisinc.com/wp-content/uploads/pn_400103.pdf
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Old 12-18-2012, 03:11 AM   #3
nfourier
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Thank you.
How come the treatment time is 50/80 sec? In the time course Covaris recommends its 2-12 min...
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Old 12-18-2012, 05:09 AM   #4
lorendarith
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Just by looking at the parameter... it is because you use just 2% duty cycles, whereas they use 5% for the desired size of 500bp. I am not familiar with what you mean Covaris recommends.

I haven't worked with the E220, only S2 but it's all similar. When I want to change the shearing size I usually just play with the time duration.

Shorter time -> bigger fragments
Longer time -> shorter fragments

I go in steps of 5-10s and that mostly gives me satisfying results. Additionally you can change the duty cycles as well, but this change is more drastic.
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Old 12-18-2012, 05:23 AM   #5
nfourier
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I read in the Covaris optimization protocol to do a time course of 2-12min and do 6 time points. This is why making changes in time duration of 5-10s seems puzzling to me...
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Old 12-18-2012, 06:35 AM   #6
lorendarith
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Hm, okay. I've always sheared my HMW DNA with similar parameters, durations of 50-80s, obtained desired insert sizes and never did these time points you mentioned; always worked fine.

Though, you wrote you do this on raw cell lines? That's probably where the difference comes from. Can't help you there, but say to just try out these other parameters and see if it works
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Old 12-18-2012, 10:14 AM   #7
Hamid
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Hi nfourier,

Most protocols and reagents used today for ChIP were developed and optimized for use with probe or bath sonicators. Since Covaris technology is quite different, different sample preparation reagents and protocol had to be developed. Based on your description, you are using a one step lysis/shearing protocol using the buffer you specified. Unfortunately one step/lysis/shearing protocols and reagents do not work with all cell lines using Covaris. The Covaris reagents and protocols are universal and will work with any mammalian cell lines and tissues. Our shearing buffer is quite simple. It contains 1mM EDTA, 10mM tris-HCl, 0.1% SDS. After shearing, you can easily add concentrated salts and detergents to the sheared chromatin to match the constituents of your IP buffer without the need to dilute.
I have attached a gel image of raw cells being processing using Covaris AFA.

Thank you

Hamid
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File Type: pdf Raw mouse monocyte chromatin shearing.pdf (193.2 KB, 73 views)
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Old 12-18-2012, 10:11 PM   #8
nfourier
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Thank you Hamid. I will try the shearing buffer. I saw in the gel that the lines are 2-10min, what does that mean? what is the exact time? and what is the duty cycle?
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Old 12-18-2012, 10:32 PM   #9
Hamid
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Hi Nfourier,

It is the lane number-processing time, so 2-10 means lane 2, 10 minute processing time.
Just using the shearing buffer might not work. You will have to use the truChIP shearing kit reagents to prepare your cells.

Thank you

Hamid
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Old 12-20-2012, 02:22 AM   #10
nfourier
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I did another run of sonications. I used Covaris shearing buffer (as suggested by Hamid) and tried 3 setups (each sample is 130ul, ~2.5*10^6 cells):
duty cycle 2%, 8min
duty cycle 5%, 8min
duty cycle 10%, 8min.

Cleaned the DNA and ran on gel. Got the same results for all 3 setups: prominant band in the 1000-3000 rangs. It seems the duty cycle doesn't change my shearing.
Any suggestions? Increase the time (with 2%duty cycle I didn't notice difference between 8 & 12min)? Increase the Peak incident power (currently at 105watts)?
I'm at loss. It shouldn't be this hard.
Gel is attached.
Thank you!
Attached Images
File Type: jpg 20.12.12 covaris 8min 2-5-10%.jpg (41.9 KB, 33 views)
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Old 12-20-2012, 08:25 AM   #11
Hamid
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Hi nfourier,

Simply increasing the processing energy will not work. It will just destroy the epitopes.
Please process your samples according to the Covaris protocol http://covarisinc.com/wp-content/uploads/pn_010145.pdf . You can contact Maria Kotler mariana@danyel.co.il to see how you could obtain a kit. There are also a few other cell preparation protocols that are compatible with Covaris AFA. Ethan Ford who contributes to seqanswers has a protocol that he uses successfully with a few different cell lines. You can search for his posts to locate a link to his protocol.
Meanwhile can you please provide the following information:
1. How do you currently fix your cells?
2. How do you prepare nuclei?
3. for DNA size range analysis do you RNase treat, and proteinase K treat your samples before loading on a gel?

Thank you

hamid
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Old 12-23-2012, 05:27 AM   #12
nfourier
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Hello Hamid,
1) I fix my cells in their medium, in formaldehyde (1.42% final concentration) for 15min in room temperature. It had worker before with 3 different cell lines in our lab.
2) I prepare the nuclei with my IP buffer (according to the FastChIP protocol): 150mM BaCl, 50mM Tris-HCl (pH 7.5), 5mM EDTAm NP-40 (0.5%), Tirton X-100 (1%). I resuspend 10^7 cells pellet in 1ml buffer and centrifuge (12K G, 1min). Resuspend again, centrifuge, and then use the same buffer as shearing buffer.
The last time I did the protocol I used the shearing buffer you suggested.
Again, this protocol has worked for us many times before.
3) For DNA analysis I do ethanol percipitation and use Chellex (as in the FastChIP protocol). Because I don't do RNase treatment I do see the RNA on my gel around the 100bp size.

I am aware of the kit, but since our protocol has worked before I am reluctant to change to a new kit.
I am familliar with Ethan's protocol. His lysis buffer is very similar to mine (a little different % in Triton X and NaCl) and his shearing buffer is like the one you recommended. He used 5% duty cycle and 12min on Covaris S2, while I did it for 8 min. While he uses TC12X12 tube I used the microTubes. Maybe that's the problem?
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Old 01-04-2013, 01:48 PM   #13
Hamid
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Hi Nfourier,

You are over-fixing your cells. I have yet to find a single mammalian cell that requires more than 5 minutes of cross linking at room temperature.
May I suggest that you follow all the steps of either the Covaris truChIP or Ethan's protocol including the fixation, nuclei isolation, and shearing. Please use methanol-free formaldehyde for fixation.
Most chromatin shearing protocols out today were developed and optimized for use with probe or bath sonicators. These protocols will very likely not work with Covaris AFA. I do understand your reluctance to change your protocol, but since Covaris AFA is a much different technology than probe and bath sonication, you will have to change your sample preparation to match the requirements of AFA.

Thank you

Hamid
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Old 01-06-2013, 02:50 AM   #14
nfourier
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Default Fixation protocol

Thank you Hamid.
I will take your advice and use Ethan's protocol. I prepared the buffers but I have a question regarding cells in suspention. Ethan's protocol has the fixation buffer volumes for adherant cells and the Covaris protocol has volumes for cells in suspention (10^8 or 2-3*10^7). I will have about 10^8 cells in suspention. How much fixation buffer do you recommand to use, 22ml (+1% formaldehyde) like in the Covaris protocol?

Thank you,
Nitsan.
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Old 03-07-2019, 05:21 PM   #15
ecbolo
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Hi All,

I'm excavating this threat because my question is somewhat related to the above.

I am using a Covaris E220 to shear highly degraded DNA (from animal hair and museum tissues). The DNA starting fragment size between 600-3,000bp and I want ending sizes to be 300bp. The official Covaris protocol to get 300bp suggests the following for DNA > 10,000bp:

Peak Power: 140 W
Duty Cycle: 10%
Cycles Per Burst:200
Treatment Time: 80s

But, I've found suggestions that the closer in size the original DNA is to the desired post-shearing size, the longer the treatment time should be: https://covaris.com/resources/faqs/#toggle-id-100. Has anyone run experiments on the treatment time needed for degraded DNA? Should I run longer than 80s? Shorter? How much of a concern is over-fragmenting?

Thank you!!!!
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Old 03-07-2019, 05:57 PM   #16
luc
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Yes, the Covaris recommendation matches our experience. You will likely need a longer sonication time. The sonication of already short fragments is comparatively inefficient.
You will have to try several durations. Perhaps 110s?
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Old 03-08-2019, 02:29 PM   #17
ecbolo
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Thank you so much, luc, this is really helpful feedback! I'll update how it goes.
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