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Hi guys. I'm working on a protocol to reduce the size of some post-ChIP DNA. I got nice sizing from 200-600 bp's in my sizing gel I ran before pulling down on my input material with my antibody of interest. However, after it seems that the epitope I am looking at is preferentially localizing to a larger fraction of chromatin (~1500 bp's). So, I'm trying to standardize a post-ChIP sonication protocol using a cup horn sonicator to get my size distribution down before I prepare some ChIP-seq libraries. Does anyone have or has heard a protocol using this sonicator that works to get a size around 250 bps? Thanks.
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We always shear our post-ChIP DNA again using a Covaris and their standard settings for the size we want. Usually we aim for 350 bp.
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What model cup horn system do you have?Comment
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chromatin shearing does generate a distribution, and the partially sheared material serve as better epitopes than the shears material. Hence you are you pulling down larger fragments that what you had expected. Depending on the energy used to shear the chromatin, your 200-600bp will likely have the epitope ripped off so they do not IP.
Consider reducing the energy/time of the shearing, and control the temperature of your samples to maintain epitope integrity.
Thank you
HamidComment
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Hi,
I'm experiencing the same problem for H3K4me3 and H3K27me3, despite that we shear the chromatin to 100-700 bp with the Covaris. The size shift is much more pronounced for H3K27me3 (aka I have a large peak >1000 bp).
We performed ChIP-qPCR on these ChIP samples as a QC before proceeding with the library preparation, and we see a satisfactory enrichment of correct histone target.
Now, I'm concerned about how this size-shift will affect the library preparation since the majority of the fragments are >1000 bp. Would it be possible to size select to remove all of those large fragments before library prep or perhaps "re-shear" the ChIP material again, before library prep?Comment
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Hi, YanMar.
I did not get what you said. When you said the size shift for H3K27me3, do you mean that the H3K27me3 can pull down the larger fragments >1000bp? In that case, if you remove the large fragments before library construction, how can you get the H3K27me3 pattern?
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