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  • Low ChIP DNA yield for histone ChIPs

    Hello,

    I am relatively new to the world of ChIPs and ChIP-seq. I am trying to prepare some samples for ChIP-seq against H3K9ac, however, my yields of ChIP DNA are low despite trying several antibodies. I am getting only about 1ng of ChIP-DNA, whereas I am told that histone ChIPs should give high yields since histone modifications are more abundant in cells. I am afraid that this might be insufficient for preparing a library of acceptable complexity. When I perform PCR using the DNA, I do see enrichment at genes of interest.

    What can I do to improve yields? I use phenol chloroform extraction to purify DNA. Since I never really see the DNA pellet, maybe I am not resolublizing my ChIP-DNA in the tube completely. Would using a column purification help me get more DNA? Any suggestions would be highly appreciated. Thanks a lot!

  • #2
    The easiest answer is to increase your sample input. I do not ChIP that mark, but when I do ChIP H3K27ac I feel comfortable with 2-3 million cells. I use Ampure XP beads to purify ChIP DNA, this works very well for me. In the past I used the columns and found that this was a less efficient than using the beads. Smaller IPs tend to be more efficient also.

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    • #3
      Hi epigenome! Thanks so much! Unfortunately, I only have the option of using phenol chloroform extraction or Qiagen columns to purify my DNA. My core facility said that they can still make good libraries with 1-5ng of ChIP DNA. However, is it common to get such low yields with histone ChIPs?

      One of my ChIPs with another histone mark gives me 100-200ng of ChIP DNA. Given this fact, I feel like I am using sufficient amount of input material for performing my ChIPs. Also my inputs show up at good Ct values when I run PCR. Thanks a lot for your suggestions!

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      • #4
        If your protocol is working fine for other marks I suspect that you have an antibody issue. I would agree with your core facility, 1-5 ng can definitely give you good libraries. Some histone marks are very abundant, as you suggest (4me3, 9me3, 36me3), while others tend to be very small (27ac, 27me3, any TF). Therefore, the size of the ChIP is not always a good indicator for a successful ChIP, although we do like to see larger quantities for logistical purposes.

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        • #5
          Thanks again epigenome! I am getting great ChIP DNA yields for K9me2 but my yields are substantially lower for K9ac. From your answer, it appears as if the methyl marks are more abundant than the acetyl marks, in which case my observations with K9ac ChIP DNA yield would make sense?

          I have tried five different antibodies and different antibody titrations per H3K9ac ChIP but my results are very similar. I am going to go with Millipore's H3K9ac antibody for my H3K9ac ChIP. Does anybody have experience using this antibody for ChIP-seq?

          Thanks a lot for your suggestions! This is helping me a lot!

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          • #6
            Hi dv88!

            I would be interested to know if your H3K9ac ChIP finally worked I have the same issue (my ChIP-seq data show no peaks for this histone mark whereas I can see nice peaks with others marks).

            Thanks!

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            • #7
              Hi vadoue,

              Although my H3K9ac ChIP yields were low, and I used low amounts of ChIP-DNA to make libraries, we did detect tens of thousands of peaks in the sequencing runs, with a high degree of reproducibility across samples.

              From other experiments, we now know that H3K9ac is not very abundant in our cell type, unlike methyl modifications that are so predominant, which could explain (at least in part) low yields.

              Also, in the past, we were able to obtain several thousand peaks (for methyl marks) by just sequencing a lot deeper, so I am not sure if this may help your case. Although I would think you can do away with fewer reads for H3K9ac since it forms nice, sharp peaks.

              Hope this helps!

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              • #8
                Hi!

                Thanks for your answer!!

                As I have no peaks only with this antibody, I am wondering if the problem could come from the antibody itself as it has been store for more than one month at 4 degres (it should has been store at -20 after max 1 to 2 weeks...)...

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                • #9
                  That might be possible. One way to test would be to do perform PCR at known, control loci with the antibody in a new ChIP experiment and/or perform PCR using the ChIP-DNA that was sequenced (if you have any aliquot left). You can also compare with ChIP-PCR results using a fresh, properly stored aliquot of the antibody. Measuring enrichment at known loci through these experiments might help answer your question.

                  Also, did you check enrichment at known loci before and after making H3K9ac ChIP libraries? Was enrichment retained after library prep? If not, it could explain why you fail to see peaks following sequencing.

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                  • #10
                    As it is a rare cell type I don't know any control loci. But ChIP-qPCR data on potential loci had always been bad with this antibody... I will try a new aliquot. Would you have any explanation of lost of enrichment after library prep?

                    Thanks for your help!!!!

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                    • #11
                      Does the cell type not express GAPDH or other very common positive control regions associated with H3K9ac?

                      If enrichment has always been bad with this antibody, that would be a problem. Perhaps you need to consider testing a different H3K9ac antibody or using a different lot number.

                      As far as my understanding goes (I can't say for sure), there are several factors that could lead to loss of enrichment during library preparation. It could be dependent on the amount of DNA you start with (low complexity libraries likely lead to low enrichment and poor sequencing results), the size of your DNA (having a high percentage of longer length fragments could be problematic, especially if you size select your libraries as you may lose diversity- and smaller fragments tend to get amplified better during PCR steps and are preferentially sequenced on the sequencer as well).

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                      • #12
                        I recommend performing an IP-Western to determine if your antibody is still good for an IP. In fact, doing an IP Western is always good practice for any experiment involving an IP. It is good to show that the antibody and IP are working as expected in your hands, even if the antibody has been "certified" for IP. And an IP-Western is also a nice piece of data to have for a manuscript.

                        Along those lines, I think using more antibody could also be a good way to improve ChIP yield, especially if paired with more input. And note that better yield does not necessarily indicate a better IP, too good of yield makes me suspect high background.

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