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Old 10-08-2010, 11:41 AM   #1
Location: Stanford, CA

Join Date: Feb 2010
Posts: 12
Default WGA Amplification for ChIP samples prior to Seq?


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!
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Old 10-08-2010, 12:08 PM   #2
Location: Kansas City

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Can you tell me what sort of bias towards GC content you are seeing?
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Old 10-08-2010, 12:46 PM   #3
Location: Stanford, CA

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Basically the GC rich sequences will be over-represented. It has been published here:
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Old 10-08-2010, 03:14 PM   #4
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Don't think they used wga but you may want to check this protocol:
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Old 10-22-2010, 08:42 AM   #5
Location: Baltimore

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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?
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Old 04-12-2012, 07:30 AM   #6
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Location: france

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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;
linampli is offline   Reply With Quote

amplification, chip-seq, low cell number

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