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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 (http://www.nature.com/nprot/journal/...t.2006.98.html), 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?
Thanks,
Kunle
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 (http://www.nature.com/nprot/journal/...t.2006.98.html), 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?
Thanks,
Kunle