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Highly Variable DNA yield from Neutrophils lysed & sheared for ChIP

shearing DNA ChIP neutrophils

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#1 emilyindallas

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Posted 03 November 2011 - 10:24 AM

Hello All,

I am using the MAGnify ChIP kit on human neutrophils, freshly isolated from peripheral blood. Because I am doing ChIP-seq, I need DNA fragments between 100-300 bp in size. The cells are cross-linked for 10 minutes in 1% formaldehyde. I am lysing and subsequently shearing 2 X 10^6 neutrophils in 100 uL of lysis buffer using a Covaris S2 for 12 minutes with the following settings:
Duty cycle : 5%
Intensity: 2
Cycles per burst: 200
Cycle time : 60 seconds
Cycles:12
Tempterature: 4C
Power Mode: Frequencing Sweeping
Degassing mode: Continuous

My problem is that while I follow the MAGnify Protocol closely, I have large variability in the yield of DNA following shearing, based on analysis on a 2% agarose gel of via analysis on an Agilent dsDNA 7500 chip. For example, a "good" shearing of 2 X 10^6 neutrophils when diluted 1:10 in TE to analyze on the 7500 chip will give a concentration in the range of 10-12 ng/uL for the 100-300 bp region. A "bad" shear run, again when diluted 1:10 in TE and run on the 7500 will give between 1-2 ng/uL in the 100-300 bp region.

Does anyone have any thoughts about why this is happening? I keep the cross-linking and shearing conditions the same between experiments. Sometimes I snap-freeze the cross-linked and lysed cells before shearing them the next day and this hasn't conclusively influenced things either way.

Any thoughts about factors that influence DNA yield from shearing and/or working with neutrophils for this experiment would be highly appreciated.

Cheers!

#2 chabraha

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Posted 04 November 2011 - 01:27 PM

Not sure if this answers your question.........but, if you have a "good shear" you'd expect a tighter base pair range of DNA fragments.......therefore your DNA concentration (or percent fragmented DNA) within your ideal window would increase (closer to 100%). On the other hand, a "bad shear" leads to a loosening of the DNA profile (wider distribution), and therefore less DNA would be present within the ideal window..........sorry if this is confusing or not what your looking for............it's Friday afternoon and I'm mentally spent. Let me know if this needs further clarification, and maybe on Monday I can come up with a better way of explaining this. Also, I am assuming you have run your sheared DNA out on a gel before......it may better help visualize what I'm talking about.
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#3 emilyindallas

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Posted 04 November 2011 - 03:03 PM

HI Chabraha,

Thanks for your response and your thoughts. I have run my samples on a 2% agarose gel and the problem with "bad" shear runs really seems to be an overall decrease in the DNA yield of all sizes throughout the smear ( the brightness of the smear on the gels i is greatly reduced). I will attach an image of a gel file showing what I am talking about with the low overall yield. On the gel, I loaded the same volume of DNA solution in each well, 10 uL of PK treated DNA solution. I think the gel illustrates what I am talking about.

I welcome input from anyone who has any ideas about variable DNA yield. I even had one experiment with the same donor where the cells that were sheared on the day of isolation gave a low yield and the cells that were frozen and sheared later gave a decent DNA yield, and vice versa.

Cheers and keep the input coming! I am very grateful.

Attached Files



#4 chabraha

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Posted 07 November 2011 - 08:31 AM

Did you RNAse treat your samples in the first few lanes?...........I was under the impression that EtBr signal under 200 was most likely RNA.
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#5 emilyindallas

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Posted 07 November 2011 - 09:27 AM

Hi Chabraha,

I did not RNAse treat any of the samples and I use GelRed as my dye for agarose gels. I also ran all of these samples on an Agilent 7500 dsDNA chip, as I mentioned above. It shouldn't pick up RNA.

#6 chabraha

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Posted 07 November 2011 - 10:01 AM

Is there a possibility that your shearing step is consistently good but that the reverse cross-link and ProK steps are inconsistent between samples? I read that you use ProK treatment before you load the DNA onto your chip/gel. Do you perform a reverse cross-link step in conjunction with the ProK treatment?
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#7 emilyindallas

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Posted 07 November 2011 - 02:12 PM

Chabraha,

I do PK treat before running on a gel but do not reverse cross-link until after I IP. The conditions and the vial of PK that I use have been pretty consistent (20 minutes at 55C per the protocol). Do you think I should reverse x-link my sample and then PK treat it? Perhaps the PK treatment is not always effective and my DNA is not running on the gel b/c it is still cross-linked to protein? Perhaps I should increase the incubation time for PK treatment of my aliquot that I used to analyze the shearing?

#8 chabraha

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Posted 07 November 2011 - 03:10 PM

yeah I think you should do the reverse cross-link step then the ProK step..........I remember posting my quick method (~3hrs) on this forum somewhere.........but it involves heating a portion of your sample up in a high-SDS buffer containing B-ME.........then adding some ProK.......then precipitating your sample, RNase treating the sample then running it on a gel.........I think I use a 1%TBE gel......................if you can't find my previously posted protocol in a timely manner PM me and I will send it to you.
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