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  • No peaks in my ChIP Seq samples

    Hello,

    I have recently received the results of my ChIP Seq data, prior to amplification I verified that the ChIP had worked using a positive control region (vs a negative control region) with qpcr. The sequencing data shows no peak in this region and actually the data looks like the input control sample (with just random reads across the genome). I am sure that the ChIP worked so there must be something wrong with the library prep or the sequencing, has anyone else had this problem?

    Thanks
    Chloe

  • #2
    Chloe, how did you detect the positive control region by qPCR? Did you compare the fold change of enrichment to the negative control region? In my oppinion, it's better to compare IP/Input% of positive and negative region, respectively.

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    • #3
      I have also had this problem in ES cells for a specific protein. I was very surprised because the ChIP worked very nicely although not as nice as in other cells where we did get loads of peaks compared to INPUT.
      In ES cells, the INPUT and ChIP samples were identical. We first thought the facility had mixed the tubes and sequenced twice the INPUT but that was not the case as they redid the sequencing and got the same result.... I am trying to optimise the ChIP for that protein in ES cells so as to get higher IP/INPUT ratio without compromising the +ve to -ve peak differences. I am working on that... Maybe there is not that much protein bound to DNA so as to be able to detect it on the sequencing paltform be yes by RT-PCR....

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      • #4
        Hi spudybudy,

        I am also facing the same problem with low expressed TF. Did you solve your problem?

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        • #5
          Hi, yes, i managed to solve the problem. it was related to the quantification. I was using less sample than I was supposed to...
          If your transcription factor is not expressed a lot or you don't expect to see that many binding sites (<1000), i would suggest to do more chips and pool them. say 8 chips or even more. Hope this helps! good luck!

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          • #6
            Hi Spudybudy
            I'm having the same problem, but not sure whether it's a quantitation, sample abundance, or some other issue. I quantitate my DNA using the Invitrogen Quant-It kit and use 5ng in my library prep. Do you have a reliable library prep protocol that addresses these problems? If so, would you mind sharing it? Thanks!

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            • #7
              Originally posted by kalidaemon View Post
              Hi Spudybudy
              I'm having the same problem, but not sure whether it's a quantitation, sample abundance, or some other issue. I quantitate my DNA using the Invitrogen Quant-It kit and use 5ng in my library prep. Do you have a reliable library prep protocol that addresses these problems? If so, would you mind sharing it? Thanks!
              Hello,

              we are having the same problem - ChIP seems to work fine (as confirmed by qPCR on selected, known bound regions), but no peaks in the ChIP-Seq data. Sequenced ChIP sample is generally looking like input - reads randomly located, no characteristic ChIP patterns, not close to genes - but sequencing quality - phred scores, number of mappable reads - are very good. Any comments would be helpful, we are currently brainstorming trying to find a cause. TIA

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              • #8
                Chloe, did you do the qPCR on the unprocessed ChIP material or on the amplified library? did you properly process the qPCR data (IP/input)? what is your enrichment in terms of foldness?

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                • #9
                  Hello there, I would like to revive this thread because the same thing happened to us now as well. ChIP-Seq data shows no distinguishable peaks, but qPCR of both the sample and the library confirm enrichment. As far as I know, these were pooled ChIP experiments.
                  Has anyone got any tips what could have gone wrong? Or is qPCR too sensitive to "confirm" enrichment? Are there any guidelines concerning Ct values? Any comment is very much appreciated! Thanks!

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                  • #10
                    I am thinking whether the library construction step has masked the peaks. Because you have to amplify the DNA using PCR, the amplification step may have bias. I guss so.

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                    • #11
                      Hi,

                      Did anyone find a solution for this issue??

                      Thanks!

                      Comment


                      • #12
                        Hi, I would like to revive this thread, I am facing similar problem! so on your TF chip-seq how many samples did you pool or how many cells per IP did you use...
                        I have this nice enrichment in my ChIP-qPCR but I get results in my ChIPseq as you guys described here in this thread...

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                        • #13
                          Is it possible that high abundancy of protein in the nucleus can cause this?

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                          • #14
                            I am facing the same problem. I see that I have excellent enrichment with % input method however chip shows no peaks. I am not sure if there is a problem with library prep or sequencing.

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