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  • Giles
    Member
    • Feb 2010
    • 39

    #1

    Multiplexing ChIP-seq

    I am aware of two Illumina kits that can do multiplexing: the "multiplexing sample preparation oligonucleotide kit" and the "TruSeq sample preparation kit". The steps between these procedures and the "ChIP-seq sample preparation kit" are qualitatively the same. However, it seems that the former two kits are designed for sequencing efforts that allow for large starting quantities of DNA (1-5ug). For ChIP-seq, you will be starting with more like 10ng.
    The question is, has anyone done multiplexing for ChIP-seq? If so, which kit did you use? And, did you tweak the final concentrations of any of the steps to prevent a massive excess of adapters during the ligation?
    Keith.
  • csquared
    Member
    • May 2008
    • 67

    #2
    Keith,

    We do it all the time with a custom kit that has some advantages to the TruSeq. Drop me a line and I can fill in the details. If you want to use the TruSeq kits, follow the protocol as written and just dilute the adaptors down 1:10 before ligation. All other steps can remain the same. The excess primer in the PCR isn't a problem.

    Shawn
    HudsonAlpha Institute for Biotechnology
    http://www.hudsonalpha.org/gsl

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    • Giles
      Member
      • Feb 2010
      • 39

      #3
      thanks for the info. That is exactly what I would have thought, but was hoping to get some confirmation.

      Comment

      • ETHANol
        Senior Member
        • Feb 2010
        • 308

        #4
        Any non-primary sequence heritable modification of genetic material. ChIP-SEQ, DNA methylation (Bisulfite-SEQ), chromatin modifications (methylation, acetylation, etc), non coding RNA.

        I've prepared several libraries using this protocol and it works great.

        csquared, I (and I bet others) would be interested in knowing the details. How about post it here.
        --------------
        Ethan

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