We have recently developed MINUTE-ChIP, a quantitative multiplexed ChIP method based on Mint-ChIP by Brad Bernstein’s lab (van Galen et al., 2016; PMID: 26687680).
Get article at Cell Reports
Access step-by-step protocol at protocols.io
Get previous manuscript at bioRxiv
Get data at GEO (GSE133056)
Our method provides a proportional measurement of true quantities as exemplified by an artificial gradient of H3K27me3 set up through mixing two samples with 100% and 0% at defined ratios. A quantitative western blot validates the mixing ratios:
The MINUTE-ChIP quantification reproduces the same quantities from the sequencing read counts:
Resulting ChIP-Seq tracks accurately reflect the true quantities:
Why is it important to use quantitative ChIP? Quantitative ChIP is true to changes in peaks and background, as exemplified by an analogy of a volcanic peak rising up above sea level (background):
Here is the boat again. For the observer, the peak now appears pretty small.
But we as an outside observer can see that it is not the volcano height that changed, it is the sea level that rose up to the peak.
Traditional ChIP normalisation assumes a constant background both on the technical and the biological level. Just like the observer in the boat who does not know the change in sea level, the method is blind to global alterations in histone modification levels.
Read the fill story at https://www.biorxiv.org/content/10.1101/557082v1