WGA Amplification for ChIP samples prior to Seq?

[angelawu]

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!

[aperera]

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

[angelawu]

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

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2862719/

[Chipper]

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

http://www.nature.com/nmeth/journal/v7/n8/full/nmeth.1478.html

[JHU-ChIPmaniac]

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?

[linampli]

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;