In looking at massive changes in the epigenome, we will sometimes do histone extractions followed by western blot looking at specific histone modifications to determine whether they change on a global scale. Usually, we then follow this analysis up with a CHIP-seq type experiment, which is where most of the information comes from. Sometimes being able to see dramatic changes in histone marks from the histone extraction is very powerful data, especially in combination with the CHIP-seq data. I was curious if anyone was familiar with a technique that would be the equivalent "non-sequencing global" assay for DNA accessibility to be done in combination with something like ATAC-seq or FAIRE-seq? In simpler terms, fill in the blank --> histone extraction : CHIP-seq :: _______ : ATAC-seq
Hi Gim,
In order to analyze the DNA accessibility on a global scale, you can use the MNase (microccocal nucleases) digestion assay. Briefly with this method, you have to isolate and digest the chromatin with MNase and then separate the nucleosomes by gel electrophoresis. Look here for more details: https://www.ncbi.nlm.nih.gov/pubmed/?term=RESEARCH+Open+Access+Pcf1%2C+a+large+subunit+of+CAF-1%2C+required+for+maintenance+of+checkpoint+kinase+Cds1+activity
Best regards
Hi Gim,
I do not have any full-proof method to propose, but rather an idea: you could perform the first steps of an ATAC-seq protocol (insertion of Illumina adapter-charged Tn5 transposase and then globally estimate differences of insertion by qPCR using primers compatible with these adapters, like when you want to quantify sequencing libraries (with the dedicated Kapa kit for example). If you don't perform any amplification after transposition and DNA purification, that should work and give you an idea of a global level of Tn5 insertion and therefore, DNA accessibility. Good luck!
One way to assess accessibility of DNA on large scale is micrococcal nuclease assay. If you need protocol contact [email protected]
Thank you for the suggestions everyone. I will update with how it turns out.
You can use DNase I hypersensitivity assay or MNase but that is not without drawbacks. Unlike RNases, DNases such as DNas I and MNase are sequence specific nucleases meaning that they recognise and cleave specific sequences which can bias your results if you use one of them. Best is to use a combination of both in your assay.
best of luck
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