Hello Community.
I am doing ChIP experiments in zebrafish.
I recently ran several samples for a ChIP after MNase digestion.
Now I run into the problem of having 60-80% of Multimappers in my mappable reads.
Does anyone have the same problems or knows why that could happen?
Some specifics:
I have "produced" this problem in 10 independent samples, plus in 4 Input samples (meaning that it is not the IP that enriches for Multimappers).
Digestion by MNase was performed to have 80% Mono-and Dinucleosomes, but no over digestion (fragments smaller than 150bp)
FastQC gives OK quality. There are some ambiguities with Kmer content, but other than that quality seems ok.
I sequenced around 50Mio reads, from which 70-80% were mappable. However, from these, 60-80% were Multimappers. When analysing the Multimappers, they are distributed throughout the genome, and all "classes" of multiplicity is present.
From that I reason that already the MNase digestion prefers repetitive sequences, but I have no idea why and how to prevent this.
Does anyone has Input on this?
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Have you previously performed ChIP with sonicated DNA in zebrafish?
Yes, I have done it, even with the very same antibodies. And no, I did not had that many Multimappers.
I find strangest that even the Input before ChIP shows multimappers
What do you mean by multimappers.
Q1: Yes, reads that map to multiple locations
Q2: Histone modification in zebrafish. Should bind to TSS, but not necessarily to repetitve regions. Either way, the Multimappers ALSO appear in the input before ChIP
Q3: Duplication rate is very low, less than 5% of all reads