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ChIP- Different DNA size for Input DNA and ChIP DNA on Agilent Bioanalyzer

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  • ChIP- Different DNA size for Input DNA and ChIP DNA on Agilent Bioanalyzer

    I ran one of my input DNA and all my ChIPd DNA on Agilent Bioanalyzer today.And I am getting different DNA sizes for my input and ChIP DNA samples.Input shows a distribution around 100-300bp while ChIp samples is distributed around 900-6000bp. I processed them together ,decrosslinked overnight and cleaned up using Qiagen PCR clean up kit. I diluted the input 360 X to get 100pg/microlitre and the ChIPd DNA samples 10X to 300-500pg/microlitre before running on the Bioanalyzer. I am using Agilent High Sensitivity kit which expired on 3rd May 2011. The ladder ran correctly. So I am not sure if this inconsistency is due to the kit.
    What could be the reason for this difference?


    Input DNA


    ChIP DNA
    Last edited by rahulr1485; 06-01-2011, 08:58 AM.

  • #2
    theoretically, your IP target binds to long pieces of shearing-resistant chromatin which are heavily under represented in your input material.
    however, i guess there are other problems. what is your IP target? what's your total yield of DNA in the IP as compared to the input amount?

    Comment


    • #3
      Originally posted by mudshark View Post
      theoretically, your IP target binds to long pieces of shearing-resistant chromatin which are heavily under represented in your input material.
      however, i guess there are other problems. what is your IP target? what's your total yield of DNA in the IP as compared to the input amount?
      My IP target is transcription factor which was found to be induced during the treatment. Concentration of IP DNA was around 1 ng/microlitre and Input was around 35 ng/microlitre. Someone suggested that there could be proteins thats slowing down the migration, so I gave proteinase K treatment, cleaned up with qiagen kit and ran it again in triplicates on the agilent. The size distribution remained the same but the concentration of the fragments were different in same samples.Another ,member of my lab had earlier carried out the same experiment and had got size distribution around 100-500 bp along with some higher bp fragments. I also followed the same protocol, but I am getting a very different size distribution.

      Comment


      • #4
        do you pre-clear your chromatin before IP? did you ever do a mock-IP + Bioanalyzer?
        another possibility for your problem is unspecific DNA fragments attaching to your beads as you appear to get a hell of a lot of DNA in your IP.

        Comment


        • #5
          Originally posted by mudshark View Post
          do you pre-clear your chromatin before IP? did you ever do a mock-IP + Bioanalyzer?
          another possibility for your problem is unspecific DNA fragments attaching to your beads as you appear to get a hell of a lot of DNA in your IP.
          Thank you for your reply. Yes, I did preclear it for 2 hours. I didn't run mock IP on bioanalyser, however, I checked it on nanodrop and it showed some negative value.

          Comment


          • #6
            Hello,
            It is nice to participate to this forum!
            I experienced the same problem...does anybody know if we can shear again the DNA after IP or if it is not recommended?
            Thank you for your help!!

            Comment


            • #7
              I've just shared a very similar issue that I encountered on my histone modifications ChIPs.

              There : http://seqanswers.com/forums/showthread.php?p=74004

              For your information.

              Comment


              • #8
                Originally posted by Pila View Post
                Hello,
                It is nice to participate to this forum!
                I experienced the same problem...does anybody know if we can shear again the DNA after IP or if it is not recommended?
                Thank you for your help!!
                you could but then your peaks won't be centered around their original position

                Comment


                • #9
                  Originally posted by Pila View Post
                  I experienced the same problem...does anybody know if we can shear again the DNA after IP or if it is not recommended?
                  I would not do that, as the identity of this peak is not clear. If you hypothesize it corresponds to unspecific binding, there is not point to spend time and money trying to do anything with it.

                  Comment


                  • #10
                    Originally posted by rahulr1485 View Post
                    What could be the reason for this difference?
                    Naive but useful question : What (precise) type of beads did you use ?

                    Comment


                    • #11
                      Originally posted by Pila View Post
                      I experienced the same problem...
                      Same problem, same question : What (precise) type of beads did you use ?

                      Comment


                      • #12
                        Hi rahulr1485 and MGineste,

                        What you are noticing is unfortunately an ideal example of epitope destruction during high energy chromatin shearing. What is occurring is that you are over-shearing your samples to the extent that you are destroying the epitope by ripping off the proteins from the DNA. High energy shearing also transfers quite a bit of heat to the sample during processing, contributing the reversing of the cross links during shearing.
                        The gel/bioanalyzer results look great, but when you IP, you only pull down what has not been sheared extensively. A very good way to confirm that is to IP different shearing time points, and check the results on the Bioanalyzer. Since MGineste is working with histones, which are highly prevalent, a western of the sheared chromatin will not be a good confirmation. if rahulr1485 is working with transcription factors, then a western will illustrate the epitope destruction during the shearing process by running aliquots of the shearing time course on a SDS PAGE for western.
                        How are you fixing your samples? and how are you shearing chromatin?

                        Comment


                        • #13
                          Originally posted by Hamid View Post
                          Hi rahulr1485 and MGineste,

                          What you are noticing is unfortunately an ideal example of epitope destruction during high energy chromatin shearing. What is occurring is that you are over-shearing your samples to the extent that you are destroying the epitope by ripping off the proteins from the DNA. High energy shearing also transfers quite a bit of heat to the sample during processing, contributing the reversing of the cross links during shearing.
                          The gel/bioanalyzer results look great, but when you IP, you only pull down what has not been sheared extensively. A very good way to confirm that is to IP different shearing time points, and check the results on the Bioanalyzer. Since MGineste is working with histones, which are highly prevalent, a western of the sheared chromatin will not be a good confirmation. if rahulr1485 is working with transcription factors, then a western will illustrate the epitope destruction during the shearing process by running aliquots of the shearing time course on a SDS PAGE for western.
                          How are you fixing your samples? and how are you shearing chromatin?
                          Mmmh... This a very technical as well as speculative explanation.

                          I may have a basic one : http://seqanswers.com/forums/showpos...10&postcount=2

                          This is why I'm insisting on the type of beads that have been used.

                          Comment


                          • #14
                            Hi MGineste,

                            Thank you for that link and data. While your theory might be plausible for people who do ChIP with carrier DNA, quite a few people including my lab, do not use carrier DNA or DNA blocked magnetic beads for obvious reasons. The "technical" explanation I gave, is based on work by us, and several other groups who have noted similar issues.

                            Thank you

                            Hamid

                            Comment


                            • #15
                              Originally posted by Hamid View Post
                              Hi MGineste,

                              Thank you for that link and data. While your theory might be plausible for people who do ChIP with carrier DNA, quite a few people including my lab, do not use carrier DNA or DNA blocked magnetic beads for obvious reasons.
                              The thing that is puzzling me at the moment is that I've been trained to ChIP in a lab very much used to ChIP-Seq approaches. They have always been using DNA blocked beads - even though we can easily foresee potential troubles using them, I agree - without encountering any issue.

                              Originally posted by Hamid View Post
                              The "technical" explanation I gave, is based on work by us, and several other groups who have noted similar issues.
                              I keep your observation in mind. Thanks for the feedback.

                              Comment

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