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ATAC-Seq large fragments

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  • ATAC-Seq large fragments

    Hi All,
    We've been trying to optimize ATAC-Seq on sorted embryonic cell populations and have run into a possible problem. After library prep we QC with a BioAnalyzer and see a large peak at around 2000bp. This doesn't change with increased amounts of enzyme or increased incubation. Any thoughts?
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    Last edited by cfuttner; 02-25-2015, 07:50 AM.

  • #2
    Originally posted by cfuttner View Post
    Hi All,
    We've been trying to optimize ATAC-Seq on sorted embryonic cell populations and have run into a possible problem. After library prep we QC with a BioAnalyzer and see a large peak at around 2000bp. This doesn't change with increased amounts of enzyme or increased incubation. Any thoughts?
    I have been using ES cells and for a while I was getting profiles like that. I started getting a greater proportion of small fragment peaks when I lowered the reaction volume to 5ul and used 2.5 ul of transposase and 2.5 ul TD. You will have to resuspend your lysed cells in the tagmentation mix. Skip the cleanup step after tagmentation. This method came from the original ATACseq paper for lower cell numbers. Hope that helps.
    Last edited by rbd; 04-02-2015, 08:24 AM.

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    • #3
      ATAC-seq and bioanalyzer

      Hi,
      I have a 'side question'- In which Bioanalyzer (and kit) did you use? I suppose you used a kit suitable for DNA library with long fragments (>1Kb)..
      Thanks

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      • #4
        Originally posted by Tal Golan View Post
        Hi,
        I have a 'side question'- In which Bioanalyzer (and kit) did you use? I suppose you used a kit suitable for DNA library with long fragments (>1Kb)..
        Thanks
        Its the Agilent 2100 Bioanalyzer using the High Sensitivity DNA kit. Its intended for fragments under 1KB but the upper marker is at 10KB so you can see some of the larger fragments as well.

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        • #5
          how many cells you used in the reaction?


          Originally posted by rbd View Post
          I have been using ES cells and for a while I was getting profiles like that. I started getting a greater proportion of small fragment peaks when I lowered the reaction volume to 5ul and used 2.5 ul of transposase and 2.5 ul TD. You will have to resuspend your lysed cells in the tagmentation mix. Skip the cleanup step after tagmentation. This method came from the original ATACseq paper for lower cell numbers. Hope that helps.

          Comment


          • #6
            Perhaphs increasing tagmentation time would help you.... also make sure the cell counts are correct

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