Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • ChIP seq DNA starting material

    Hello,
    I'm new in the ChIP-seq field and had issues with my first experiment. I started with 5 ng of ChIP-DNA (as measured with Qubit, bioanalyser gave slightly different results)then followed the Illumina protocol with 18 cycles of PCR. When the sequence went back, I found >95% of duplication. The only peaks identified by macs seem to be PCR artifact. The antibody I used was validated by western blot and mass spec analysis after immunoprecipitation. So I know that the IP step is working.

    I was considering re-doing the experiment increasing the amount of DNA to start with and maybe decreasing the number of PCR cycles.

    Illumina recommends 5<<10 ng of DNA as starting material. Does anybody tried to start with more ie 30-50 ng? Looks to me as the only way to get better results

    any help will be greatly appreciated.
    many thanks
    Olivier

  • #2
    Did the bioanalyzer show the ChIP-DNA in a good size range (~100-700bp)?

    Personally I don't understand why Illumina's ChipSeq kit uses an agarose gel band extraction. Even if you are pretty good at extracting DNA from agarose, it is good way to lose >90% of the DNA.

    --
    Phillip

    Comment


    • #3
      Hi Phillip,
      thank you for your answer
      yes the size of the fragment was fine according to the bioanalyser.
      Did you succesfully run Chip seq without the gel extraction step?

      thanks
      Olivier

      Comment


      • #4
        Any chance you could post the bioanalyzer chromatogram of your input DNA? I ask because we always run this as QC, but I never see example chromatograms in ChipSeq kit manuals of this step. (They always have a picture of the library, after PCR amplification, instead.)

        We switched to the Bioo ChipSeq kit -- it doesn't use a gel extraction kit. I think that as a result of this it is able to specify 1 ng of input DNA.

        That said, the libraries we made from the Illumina ChIPseq kit starting with 5ng of DNA were in the 40-60% duplicate read range. We did once make a set of libraries that had a similar level of duplication to yours -- but in most cases those started with an amount of DNA below the level of detection of the Qubit. (For one ul -- if we had used the entire sample, for fluorimetry we may have been able to detect its concentration. But there was no point doing that...)

        --
        Phillip

        Comment


        • #5
          bioanalyser data here:

          Comment


          • #6
            sorry, the previous link is not working.
            here:http://imgur.com/a/CRUzA

            Comment


            • #7
              Originally posted by oliPap View Post
              sorry, the previous link is not working.
              here:http://imgur.com/a/CRUzA
              That is input? Wow, that looks great.

              --
              Phillip

              Comment

              Latest Articles

              Collapse

              • seqadmin
                Strategies for Sequencing Challenging Samples
                by seqadmin


                Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                03-22-2024, 06:39 AM
              • seqadmin
                Techniques and Challenges in Conservation Genomics
                by seqadmin



                The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                Avian Conservation
                Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                03-08-2024, 10:41 AM

              ad_right_rmr

              Collapse

              News

              Collapse

              Topics Statistics Last Post
              Started by seqadmin, Yesterday, 06:37 PM
              0 responses
              8 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, Yesterday, 06:07 PM
              0 responses
              8 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 03-22-2024, 10:03 AM
              0 responses
              49 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 03-21-2024, 07:32 AM
              0 responses
              67 views
              0 likes
              Last Post seqadmin  
              Working...
              X