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  • WGA Amplification for ChIP samples prior to Seq?

    Hi,

    I am working with small cell number ChIP-seq, and was wondering if anyone has any experience doing a WGA or similar after the ChIP step, before going on to make the library and seq?

    I have tried doing the Illumina PCR amplification protocol after attaching the adapters, but it results in a lot of bias based on size and GC content, which for small samples basically kills the specificity of the experiment.

    I'm considering the WGA kit from Sigma... any experience/guesses on whether that might work better? I'm also open to other kits people have used.

    Thanks in advance!

  • #2
    Can you tell me what sort of bias towards GC content you are seeing?

    Comment


    • #3
      Basically the GC rich sequences will be over-represented. It has been published here:

      Comment


      • #4
        Don't think they used wga but you may want to check this protocol:

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        • #5
          I don't have any experience with small cell numbers. Are you pulling down a histone modification or a TF? The only publications I have seen using low cell numbers (10,000 cells) and successful ChIP-seq were using antibodies against histones. Has anyone sequenced a ChIP-seq library from a TF and low cell numbers?

          Comment


          • #6
            check this out...
            Single-tube linear DNA amplification for genome-wide studies using a few thousand cells.
            Shankaranarayanan P, Mendoza-Parra MA, van Gool W, Trindade LM, Gronemeyer H.
            Nat Protoc. 2012 Jan 26;7(2):328-38. doi: 10.1038/nprot.2011.447.
            Nat Methods. 2011 Jun 5;

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