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X-linking, Shearing and ChIP with the Covaris S2

December 28, 2011

I decided to look at how crosslinking conditions affect ChIP for a histone variant I am working on. I used three crosslinking conditions and shearing times which would shear the chromatin well. Each ChIP was from one 15 cm plate of HeLa cells and cells were processed according to my Covaris ChIP protocol and the amount of DNA recovered was quantitated by the QuantIt HS DNA kit from Invitrogen. The crosslinking/shearing conditions and the amount of DNA recovered are listed below:

1) Fixed 1% formaldehyde for 5 min in my ‘Covaris Fixing Buffer’ and sheared for 5 min. 7.7 ng of DNA recovered.
2) Fixed with 1% formaldehyde for 10 min in my ‘Covaris Fixing Buffer’ and sehared for 20 min. 7.5 ng of DNA recovered.
3) Fixed with 1% formaldehyde added directly to the growth media while it was still warm from the incubator and sheared for 20 min. 10.4 ng of DNA recovered.

If you look a couple posts back when I looked at crosslinking conditions, shearing times and effects on the epitope by Western blot it appears there is a huge loss of epitope when the cells were crosslinked in media. There is also a significant reduction of epitope when the shearing goes longer than 10 min. Thus, I found it interesting that I recovered more DNA from the sample crosslinked for 10 min in growth media. My guess is that more of the protein is getting crosslinked to the DNA so the IP is more efficient despite the loss of epitope from crosslinking and shearing. The could also be a result each eiptope bringing down more than one piece of DNA due to the higher degree of crosslinking.

Another interesting thing is the qPCR of the ChIP. I looked at a couple loci where this histone variant does not bind (the two on the left) and five loci where it does bind (the five on the right). I know this from a previous ChIP-seq experiment. It appears that the more the crosslinking the better the ChIP performed. Given that this is a histone variant and you don’t get proteins that are much more tightly associated with the DNA than histones, I would guess that this observation would hold for many other proteins. Crosslinking for 5 mins in ‘Covaris Fixing Buffer’ may result in better shearing but that does not necessarily correlate with how well the experiment works.

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15 Comments
  1. Hi Ethan,
    I know you have a great protocol for Covaris sanitation…but I was wondering whether you had a chance to test truChIP High Cell Chromatin Shearing Kit from Covaris.
    Please let me know.
    Thanks!
    TK

  2. sonication not sanitation…;-)

  3. ethanomics permalink

    Hi TK,
    The protocol I have is essentially the same as the truChIP High Cell Chromatin Shearing Kit from Covaris. There may be some slight differences in buffer composition but nothing that will make much of a difference. That being said I used the Covaris kit for a couple preps and the shearing looked the same. If you use the Covaris kit, what is important is to add Triton X-100 to the chromatin after shearing. Triton X-100 sequesters the SDS which improves antibody binding (other non-ionic detergents can do this as well).

    The truChIP kit is great product in that it gives people a easy straight forward method to shear x-linked chromatin for ChIP using the Covaris machine. Without it people would be using protocols that don’t work and using tubes that don’t work and be disappointed with the performance of the machine. But for people that are wiling to look around, your better off using my protocol because the you know what you are doing.

    Ethan

  4. Thank you Ethan!
    TK

  5. Sorry..may I ask one more question?
    There are two kinds of truChIP (Non-ionic Shearing Buffer
    and SDS Shearing Buffer). Have you tried both?
    Thank you,
    TK

  6. ethanomics permalink

    I have only tried the SDS shearing buffer. I would not recommend using the non-ionic shearing buffer. It is important to open up the chromatin with SDS while shearing to reduce biases caused by chromatin structure. 0.1% SDS is enough to do this. Then you can add a non-ionic detergent such as Triton X-100 to sequester the SDS for antibody binding. It is the best of both worlds. I think this is clearly the best way to go.

    On the truChIP kit, you can see exactly what is in the buffers in the MDSS listed on the Covaris website. Covaris uses PIPES in the ‘Lysis Buffer’. I use HEPES. The detergent is a little different but not something that matters.

  7. Thanks again!
    TK

  8. SLEE permalink

    Hi, Ethan

    We’d like to perform the pilot experiment for optimizing the condition for cross linking/shearing time with primary mouse cells. Of course, Covaris will be used for sonication. Do you have any idea about primary cells for cross linking/shearing or difference from cell-line? If so, please give us any advice.
    it would be a great help…

    Thanks..
    SL

  9. ethanomics permalink

    Hi SL,
    In my experience we don’t see much of a difference between HeLa, mouse ES cells and MEF’s. I think the bigger consideration is what is the optimal X-linking for your protein. To that end this is what I would recommend: X-link with the following conditions:
    1)1% formaldehyde 5 min (shear 12 min)
    2) 1% formaldehyde 10 min (shear 25 min)
    3) 2 mM DSG for 45 min followed by 1% formaldehyde for 5 min (shear 15 min)

    Then for each x-linking condition do a shearing time course of 10 min, 20 min and 30 min.

    If you don’t want to do all that work you could just go for the following shearing times in the parentheses. And if you just want to give one condition a quick run through just pick one of the above conditions.

    Ethan

  10. SLEE permalink

    Thanks. Ethan ^.^

    SL

  11. Robbie Sierra permalink

    It seems as thought they took down the MSDS link. Would you happen to have the sonication buffer recipe?

  12. ethanomics permalink

    Click on the “protocols” tab up at the top and look at my Covaris ChIP protocol, which is here for now:
    https://ethanomics.wordpress.com/chip-fragmentation-with-covaris-protocol/

    It’s something pretty close to that. Regardless what I have written in the protocol works. One thing Covaris missed is that the SDS need to be sequestered with Triton X-100 before you do your IP for most antibodies.

    • Robbie Sierra permalink

      Awesome thanks. I had one other question. In your protocol you say your using six 15 cm plates, are those plates combined into one 15 ml conical tube towards the end of “X-link cells” or do you prepare each plate separately? Do you use the same 10 ml of PBS to scrape each plate?

      • It sort of depends on the size of the cell pellet but one 15 cm tube should be big enough for six 15 cm plates of HeLa’s. Two might be better and you would have a balance.

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