I am performing ChIP-Seq experiment for REST, H3K9Ac and H3K9Me2 marks in rat cortical tissues. I successfully made the ChIP libraries. At this point, I am wondering about the sequencing depth for these marks. I believe that H3K9Me2 and H3K9Ac are broad-source factors whereas REST is a point-source factor. Hence I am planning to go for 40M reads for histone marks whereas 20M reads for REST. Please advice me on this if anyone has the prior experience on these marks.
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Those are good starting points from my experience with TF markers. I would sequence what you have, reserving some sample if needed for more depth later, as well as confirmation of any sites identified using qPCR.
Hi Anil,
I agree with William for REST. H3K9me2 indeed covers broad regions. My experience with acetylation of H3 is that it is somewhat in between: mostly small domains of a few kbs. 40M should be largely enough though.
If you want to do reliable peak calling, you SHOULD include sequencing of your input too. This doesn't need to be sequenced very deep (about 20M reads is enough), but is essential to exclude random peaks caused by sequencing artifacts. If your 3 ChIP-samples are from the same cell type (and preferably chromatin preparation), you only need to do this once.
Finally, keep in mind that doing replicates strongly improves the confidence of your identified peaks. Best is to do biological replicates, but this means doing the entire experiment (including sequencing) twice.
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