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  • ChIP-seq problem

    I want to use ChIP-seq to find the new target sites of a transcription factor.but i was confused by the sample preparation:excise 100-200bp
    dna fragment from gel after ligation and pcr to sequence.
    in ChIP,the DNA was sonicated into 500-1000bp,so where were the 100-200bp ragments from?

  • #2
    In the sample prep, people usually collect short fragments (eg 100-200bp length). The shorter the fragment, the better the resolution you'll have during the analysis. In your experiment, you've collected MUCH larger fragments, so your resolution will be about the same as a ChIP-Chip experiment.

    At the risk of sounding silly, whatever size you collect is where the fragments come from.
    The more you know, the more you know you don't know. —Aristotle

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    • #3
      I think what musicflower feel puzzled is that majority of sonicated dna size is not consistent with dna fragments selected on gel. I would say this is a trade off of getting as much sonicated dna with small size and not damaging protein-chromatin interaction too harvard by sonication.

      so even though majority of sonicated dna are within 500-1000bp, the ones that are actually useful for subsequent steps are those that are very small (<500bp). For this reason, it doesn't seem too contradictory.

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      • #4
        ChIP Seq

        Has anybody tried to perform ChIP-Seq using 454? If yes then how much amount of ChIP DNA you have started? and what was the size range? How was final result? I am keen to know any thing about ChIP-seq experiment using 454.

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        • #5
          Just as a general comment - and it may not answer your question, 454 is the least used for ChIP-Seq because you don't need the longer reads, and you do need as many fragments as possible (up to the point of saturation.) Thus, you're really not picking the right equipment if you're using 454 for ChIP-Seq. You're probably better off getting more (and shorter) fragments using SOLiD or Illumina for your average chip-seq experiment.
          The more you know, the more you know you don't know. —Aristotle

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          • #6
            I totally agree. Think there is no specific paper either for 454-ChipSeq
            --
            bioinfosm

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            • #7
              ChIP Seq

              Since. we have a transcription factor which binds in repetitive region (in most of the cases), so we have to have higher read length(350-400 bases)in order to map it. Now, it is only possible with 454. Else what could we do?

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              • #8
                Originally posted by seqexpert View Post
                Since. we have a transcription factor which binds in repetitive region (in most of the cases), so we have to have higher read length(350-400 bases)in order to map it. Now, it is only possible with 454. Else what could we do?
                454 might work if you have a small genome, else you coul do paired-end to increase mappability. What type of repeat is it?

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                • #9
                  These are basically SINE repeats.

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                  • #10
                    Problem of ChIP-seq

                    When I enrich my samples after ChIP by adapter-amplified manner, I face an abnormal DNA amount increase of 1% total input samples, which is almost 10000 fold higher than ChIP one. (Original ration of 1% Total input to ChIP one is 1 to 1.2.)

                    Ex: Original DNA amount of 1% total input sample: 50ng
                    Original DNA amount of ChIP one sample: 60ng

                    DNA amount of 1% total input sample after amplification: 30,000,000ng
                    DNA amount of ChIP sample after amplification: 3,000ng

                    *Sample preparation by Millipore ChIP Kit
                    *ChIP-seq system: SOLiD

                    Does anyone get this problem before or have some suggestion of what may cause this kind of problem?

                    Thanks a lot!

                    YoHua Li

                    Comment

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