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  • MeDIP-Seq (Illumina/Solexa)

    Dear All,

    Does anyone of you have experience with MeDIP-seq? We are currently trying to do this sequencing using Illumina/ Solexa technology, but we couldn´t get enough reads.
    Normally we get around 15 million reads, but for our sample the maximum that we could get is 700 000.

    Do we need some special parameters during the cluster generations for the MeDIP sample? for example adding betain or increasing the T° of the denaturation before the first sequencing cycle?

    I will be very grateful if someone share his experience with us.

    Thanks in advance
    Regards

  • #2
    HEllo,
    I am also interested in doing MeDIP-seq. Do you have a working protocol where I can try out.

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    • #3
      We've done quite a bit of MeDIP and related types of study. The MeDIP libraries get treated much like any other ChIP library. Their composition is slightly biased but not to a level where it causes problems with library generation or sequencing.

      The main issue we've seen has been duplicated libraries resulting from over-amplification of the ChIPped material. In many cases this was because we were looking at libraries for samples with virtually no methylation, so very little material came out of the ChIP. Once the libraries have been prepared and quantitated we've had very good yields out of them though. If you're having a problem and other types of library are working OK then I'd suspect the ChIP protocols first.

      You can see the protocols used on our site in one of our recent publications (the supplementary info will contain much more useful information). If you have specific questions I can ask the people who actually do the sample prep.

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      • #4
        The main difference between MeDIP and ChIP is that after the IP, MeDIP DNA is single-stranded, whereas ChIP DNA is double-stranded.

        You have a few options (off the top of my head):
        a) add the Illumina adaptors before you do the MeDIP
        b) regenerate the second strand of the MeDIP product before putting on the Illumina adaptors
        c) some other tricky stuff

        So to avoid all these problems we decided to use MethylMiner from Invitrogen, which keeps the DNA double-stranded.

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        • #5
          Has anyone tried using MethylMiner to enrich DNA that has been tagged and fragmented by Nextera? I would like to try this protocol, but it would be nice to hear others input before potentially wasting the time and money.

          For example: Nextera tagmentation --> MethylMiner --> TruSeq/Nextera probe hybridization enrichment --> Sequencing of native DNA and methylated DNA fractions.

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          • #6
            Nextera MeDip !!! hell yeah

            this sounds like the best option for me??? Did you try it out I would like to try it as well (I'm a big Nextera fan)!!! I read somewhere else that Illumina primers can be methylated sometimes, but what about the transposon insert from the Nextera? If the nextera transposon adapters have a methylated site that would certainly would be a problem.
            Last edited by aleferna; 07-06-2015, 01:39 AM.

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