Hello,
We are attempting to perform ChIPac-seq, a method to look for histone tails which have been proteolytically cleaved. This involves crosslinking, isolating the chromatin, acetylating all lysines residues using acetic anhydride, and then performing ChIP for H3K14ac. We know the proteolysis of H3 N terminus occurs after the 18th amino acid. For control, we use a H3 C terminus antibody and look for regions of depleted signal in the resulting ChIP-seq reads. I have performed ChIP-seq on chromatin from C2C12 cells for H3K14ac and H3 using 50 ug's of chromatin and 10 ug's of antibody. In my subsequent data analysis, I was expecting to see a nucleosome depleted region at transcription start sites when looking at H3K14ac and H3 signal around TSSs. I do not see this, and have no idea why. I was wondering if anyone could provide help and if this could possibly be a bioinformatic issue?
We performed paired end 150bp sequencing and achieved upwards of 30 million reads per sample, and get good genome wide coverage. Data was aligned using bowtie 2, filtered and converted to sam/bam with sam tools, and duplicates removed with picard. Deeptools was used to make bigwig files as well as heatmaps (Attaching one below as an example).
Any thoughts or advice as to why we don't see a nucleosome depleted region as essentially in both cases we are performing a ChIP for all histones?
Thanks, Ben
You can't see nucleosome depleted region and +1 nucleosome (the peak in your plot doesn't look like +1 nucleosome since it's too wide) in both H3K14ac and H3 ChIP-seq data? It's quite strange since ChIP-seq on H3 should show nearly all nucleosomes, but you can only see one wide peak in such a big area (about 8kbp).
How about CTCF binding site, where you should see a small NDR and a well-phased nucleosome array?