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Old 03-07-2017, 03:14 PM   #1
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Default ChIP-seq - all QC looks fine, but low number of peaks with sequencing

Hi all,

I've been struggling with some ChIP-seq experiments for a while now, and I'm wondering if there's something obvious I'm missing. I've been trying to ChIP Med12 (mediator subunit), Rad21 (cohesin subunit), and CTCF in a few different mouse cell types (including ESCs), using antibodies that have been used for ChIPs in multiple other studies. My fundamental issue is that I get ChIP samples, and then libraries, that look fine by every metric I can assay (qPCR enrichment >5-fold, in some cases >25-fold, WCE traces and completed library traces by BioAnalyzer with expected distribution, normal alignment and complexity once sequencing comes back) but I get very few peaks (<50 for Med12, <5000 for Rad21 and CTCF, etc.).

As another data point, my IPs have been around 5-20ng (as quantified by BioA), seemingly regardless of cell number. I've been using basically this protocol from the Young lab (, with the modification of different wash buffers (lo salt sonication buffer, hi salt, LiCl, then TE + NaCl)

Looking at the tracks, where I would expect to see peaks I just see a lot of noise. I have tried sonication by Bioruptor and Covaris, scaling up the number of cells from 10 million/IP to ~40 million/IP, and various other tweaks, to no avail. I'm wondering if this is a problem that other people here have faced and perhaps I'm missing something obvious?

kdemuren11 is offline   Reply With Quote

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