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  • #16
    Hi Jubs and others,

    thanks for making .pdf of the protocol available in your earlier post. I am just preparing to do try this protocol soon.. and after seeing mtDNA comments I started thinking whether a very mild lysis, followed by a quick Dounce "homogenisation" and then wash would help to get rid of mitochondria.. Or alternatively Dounce "homogenisation" and wash after the tagmentation reaction, before DNA isolation, to get rid of mitochondria with tagmented DNA

    Oh and btw, did you lyse on ice for 10 minutes or how long? (in whatever concentration of IGEPAL 0.1 or 0.01)

    Cheers

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    • #17
      When you looked at the original data how many reads did you get aligning? I am only getting ~50% and I was wondering if that is what others are getting.

      Thanks in Advance!

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      • #18
        Hi. Just trying out the ATAC-seq protocol in its original form. Any tips on how to not lose the nuclei when pipetting out the supernatant? If i carryover residual lysis buffer, I'm afraid I might see more of the mitochondrial DNA contamination you all are talking about.. Any tips?
        Last edited by SamanthaC; 01-11-2017, 07:07 PM.

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        • #19
          Originally posted by Jubs View Post
          Hi Jimmie,

          No, I have only sequenced the library that gave best results. I agree it would be nice to have technical replicates with the different detergent concentrations, but, you know, money... :P

          I measured mtDNA by qPCR, and the only 'useful' information I have is the obvious: more detergent, less mtDNA (plus, with no detergent at all, mtDNA goes up to the roof).

          And in agreement with what I said in my previous reply, the more mtDNA, the higher the enrichments of regions of interest. So, in samples with high mtDNA, the fold enrichments of regions of interest are higher than in samples with less mtDNA.

          Cheers!
          Hi Jubs,

          How did you measure the mtDNA content with qPCR. Was it an absolute measurement or a relative comparison? I've been thinking of doing a qPCR for a housekeeping gDNA gene and a mtDNA gene and comparing the relative expression. But will this give me a true indication of the % of mtDNA I have?

          Nick

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          • #20
            Hello everybody,

            Happy to have found this thread and here I have a question:

            The more detergent during the ATAC preparation, the less mtDNA, any difference as to what detergent? NP40 vs Tween for example?


            J

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            • #21
              Hi guys
              I saw a protocol on other lab where they use 2% Digitonin as a detergent.
              they said mtDNA significantly reduced by using this detergent.
              Hope it works with you guys

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