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ChIP (fragmentation with Covaris) Protocol

ChIP (fragmentation with Covaris) Protocol

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19 Comments
  1. Serge permalink

    Hey Ethan,
    Thanks for all the protocols! Very well detailed. I’m trying the ChIP protocol for the first time, using the Covaris for shearing. Then going to do multiplexing.

    Small question about you ChIP protocol. The 2 um filters that you are using in your protocol, their from what compagny? Have a hard time finding them….

    Thanks!
    Serge

  2. ethanomics permalink

    Hi Serge,
    I’m not sure if the filtration makes much of a difference. I started doing it when I was using a tip sonicator and there was some insoluble material that wouldn’t spin down and was causing an increase in the background. With this Covaris protocol I don’t see as much of it in the tube, but I figure it couldn’t hurt so I left it in the protocol. Any syringe filter should work, but try to find one that is rated ‘low protein binding’. I am using these from Sarstedt
    http://www.sarstedt.com/pdf/katalog/en/SARSTEDT_E_0409%2099.pdf
    catalog number 83.1826.001
    Good luck,
    Ethan

    • Serge permalink

      Thanks Ethan,
      I was looking for 2 um por size filters like mentionned in the protocol…..not 0.2um. Typo I guess!

      It was my first time with the Covaris S220, and did not shear my ES cell chromatin in small enough fragments. Samples were sent from collaborators and might have more cells than I tought. Doing it today again with more intensity to get in the 200-700 range.

  3. Kathleen Clark permalink

    Hi Ethan,

    I am new to ChIPs, but have been trying to optimize our lab’s ChIP protocol for use with the Covaris S2 instrument. My experience so far has been that sometimes it works, and sometimes it doesn’t, based on qPCR results of known targets for ETS1. We get good shearing, ranging between 200-600 bp, and a good yield of ChIP’d DNA, usually ~15-20 ng from 3 million cells. We essentially follow the protocol you have on your site, at least from the beginning until after shearing. There are two big differences which may be affecting our ChIPs – we use the 12X24 tubes with 300 ┬Ál, and shear for 5 minutes at 20% duty cycle and 8 intensity. So, while our protein seems to be preserved, based on W.blots, I wonder if we are too harsh? Since we still IP a lot of DNA, I don’t know if this really is the issue. Since seeing your website, I think I’ll try the other tubes and your shearing parameters to see if that makes a difference.

  4. ethanomics permalink

    Hi Kathleen,
    The 12×24 tubes are definitely a problem. All the energy is going to go into moving the sample around the tube and not shearing. My guess (a guess and only a guess) is that you are not shearing the DNA as well as you think and the DNA looks a lot smaller then in really is because you are overloading the gel. When you overload a gel the DNA runs a lot smaller then it really is. I know this does not make sense but it is absolutely true. I had zero luck with getting shearing with the 12×24 tubes and everything worked great when I switched to the 12×12 tubes with the AFA fiber. The difference was night and day.

    So my suggestions are: 1) Get the 12×12 tubes and the appropriate tube holder. 2) Load a titration of reversed x-linked DNA on an agarose gel to find the minimum amount. Run you agarose gels the full length of the gel. That will tell you the true size of your fragments.

    Take a look at page 11 of this protocol. As more DNA is loaded the fragmentation looks better and better. Of course they completely get the interpretation of the gel wrong. Regardless, it is clear that overloading makes it look like the DNA fragmentation is a lot better then it really is.
    http://myers.hudsonalpha.org/documents/Myers%20Lab%20ChIP-seq%20Protocol%20v041610.pdf

    Ethan

  5. lesande permalink

    Hi Ethan,
    there are 2 options for TC 12×12 tubes from covaris–one has AFA and costs more while the other does not have AFA. Have you ever compared the TC12x12 with and without AFA? Apparently the AFA results in better sonication but so far I have only tried the one with AFA and wanted to save money by going with the one without AFA but have not tried that yet.
    Thanks
    Les

    • ethanomics permalink

      Hi Les,
      I was told by Hamid at Covaris that the AFA fiber is necessary. It keeps the liquid from moving around the tube. The idea is that without it, most of the sonication energy is spent moving the liquid around the tube. Whereas, with the AFA fiber the energy is used to fragment the chromatin.

      But I never tired it without the fiber. If you wanted to save money, I’d suggest reusing the tubes. But keep an eye on them because they will break after a while.

      Ethan

  6. Rebecca Gentek permalink

    Hi Ethan (and others),

    compliments on your page and the protocols that go with it! I have a quick question concerning chromatin shearing on the Covaris, using the AFA tubes/system: What is the maximum # of cells (well, chromatin equivalent) you have successfully used to shear in the 6×6 or 12x12mm tubes, respectively? I wonder whether it is possible to increase the concentration beyond 1e7 (that’s the maximum I found in most protocols: my facility is only performing shearing of naked DNA and cannot help me further). Alternatively, what are people’s experiences with shearing a single sample in multiple tubes and pooling afterwards? Given the reliability of the AFA shearing, that should be ok, too, but of course sample handling/incubation times on ice etc. will be different between the different aliquots.

    Any help/suggestions would be very welcome! Thanks in advance!

    Cheers,
    Rebecca

    • ethanomics permalink

      Hi Rebecca,
      The most cells I’ve sheared in one TC12x12 tube is one super confluent plate of HeLa cells. I’m not sure how many cells that is. I think it is fine, if you have more cells to just pool. That is what I have done in the past. You can re-use the TC12x12 tube for different aliquots of the same sample if you are going to pool them.

      The only other thing I would add is adding Triton X-100 to your sample to sequester the SDS is really important. I’m saying this because I haven’t seen anyone else doing it so it might not seem necessary, but keep in mind their shearing buffers are different.

      Let me know how it goes.

      Ethan

  7. Rebecca Gentek permalink

    Hi Ethan,

    wow, thanks for the quick response! You suggest adding Triton x-100 to the lysis buffer, in which the sonication is carried out? My ChIP protocol states adding it at 1% final conc., but only for the actual IP, i.e. after the lysed chromatin is sheared.

    Cheers & thanks,
    Rebecca

  8. ethanomics permalink

    Yes, after the chromatin is sheared. The SDS opens up the chromatin for shearing and then the Triton X-100 sequesters the SDS so your antibody will work. A trick I learned from 3C protocols. You’re IP probably won’t work in the shearing buffer, which is (was) the same as the shearing buffer in the Covaris TruChIP kit but they don’t tell you that, which is why kits where you don’t know what the reagents are can sometimes be bad.

  9. Rebecca Gentek permalink

    Great, that’s what I am doing anyhow! (I am using home-made buffers) It’s good to know, though, why that’s useful. Thanks, again!

  10. ashley permalink

    Hi Ethan,
    I was wondering if you have tried or could suggest a sonication method using the covaris S2? I have 1×10^7 lymphoblastoid cells that have been cross-linked for 10 min in 1% methanol containing formaldehyde. I have the smaller 6×16 covaris tubes and I am wondering what would be the best (duty cycle, intensity, cycles/burst, min) to get DNA shearing around 200bp? I have tried d.c 10%, int=5, c/b=200, for 8min and that got the DNA to the right size but my chip didn’t work so I don’t know if I am sonicating too much.

    Thanks for any help!
    Ashley

    • ethanomics permalink

      Hi Ashley,
      Sorry a for the delay, too busy as usual. I wish I had a good answer other then ChIP can be a real pain and sometimes you have to try a few protocols. Sonicating too much can definitely be a problem with some epitopes. You might try a micrococcal nuclease (I have one posted) based protocol if your epitope is prone to being sheared off the DNA. Or you could try reducing the shearing time. The things you could try are endless (unfortunately). Wish I could help more. ChIP can be such a problematic assay.

      Ethan

  11. ashley permalink

    Ok, thanks.

  12. Rebecca Gentek permalink

    Hi there,

    same here, sorry for the delay. Time to ‘pay back’ for me:

    I have sheared chromatin from primary murine T-cell son the covaris. These are the ‘master’ settings I have used:

    Duty Cycle 2%
    Intensity 3
    200 cycles/burst
    power mode frequency: sweeping
    Continous degassing
    H2O temperature 5C (wel, the lowest the machine/computer will allow you to)
    1 cycle: 30s on + 30s off

    Unfortunately, as mentioned above, our facility does not have the 12mm holder (and tubes). Therefore, I set out testing the maximum amount for the 6mm (AFA fibre) tubes which still gave good shearing.

    I also played around with the amount of cycles, here is what I tested, for you to have an idea:

    # of cells/tube (in a volume of 130ul, i.e. the maximum volume for these tubes):
    1/3/5/10/15e6

    # cycles:
    10 – 18

    These settings obviously crucially depend on your cells and also buffer conditions (in my hands, the % of SDS in the chromatin lysis buffer did make a difference for the shearing), but these might be good starting points.

    Eventually, I did:
    chromatin equivalent of 10e6 murine T-cells/tube
    16 cycle (of the above conditions)

    yielded in 35-40% fragments of 100-250bp (which was my target size range).

    Hope that helps?

    Cheers,
    Rebecca

    PS: You’re right, Ethan, it oh-so-straightforward but still sucks.. But then, it’s just a pretty cool technique =)

  13. Hi Ethan,

    I am trying to optimize a ChIP protocol for our lab. I read your protocol and i have some questions:
    1- are the volumes listed for lysis, wash and shearing buffers for 1 (15 cm) TC plates or for pooled 6 plates?

    Thanks ahead

    • ethanomics permalink

      It is in theory for 1 x 15 cm plate but I use the same volumes for 1 x 10 cm plate.

  14. Thanks a lot Ethan !

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