We aim to verify ATAC Seq data by CHIP-qPCR of the ROIs and are currently establishing all the controls.
We use anti-H3K27me3 and H3K27Ac to pull down our histone marks of interest.
We also used IgG and H3K4me3 as IP pos and neg IP controls.
For PCR controls of all samples we use GAPDH TSS primers as positive ctrl. for H3K27Ac and H3K4me precipitated samples and as negative control for IgG and H3K27me3 (worked well); MYT1 Primers as positive ctrl. for for H3K27me3 (also worked well) but we still lack a proper negative control for H3K27Ac.
Here, we tried primers for several gene deserts (from Active Motif as well as self-designed one) which all turned out to give Ct values as low as those of positive controls. Since we can finally not distinguish whether the H3K27Ac antibody is unspecific or our PCR primers do not bind specifically, I'm trying to find somebody, who has a proper working PCR negative control for H3K27Ac precipitated CHIP-samples and might provide the sequence/reference or maybe could recommend a good working antibody (as this might be an alternative source of error). We used antiH3K27ac – Abcam ab4729 so far.
Thank you!
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