Hello , I have used TBP protein antibody to immunoprecipitate between 2 conditions : 1 - RNA interferece for TBP (Treatment) 2- Without interference (control) my experiment showing in Treatment condition TBP is not binding and in control TBP is binding. I want to prove this by chipseq analysis. I performed Chipseq analysis using Bowtie for alignment , MACS2 for peak call and macs2 bdgdiff for differential peak beween control and treatment , i got 3 peak files common , treatment over control , control over treatment . My question is , how can prove in Treatment TBP is not binding and in control TBP is binding . What next method i have to use ? I used MEME-chip and Rsat to find TATA region but couldn't found in both treatment and control . can any expert suggest ?
The difference between your RNAi and control sample should be large enough that showing an average plot and heatmap centered around TSSs, from a program such as deepTools, should show a clear loss of signal in the KD. That along with a western for validation of the KD would be convincing evidence. Since you've already done peak calling and differential peak calling as well, you could describe the number of peaks and differential peaks in both samples. I can't imagine you will be getting many strong peaks in the KD relative to the untreated if your KD worked.
@rpolicastr Thank you , for suggestions and i got answers
Do you have experimental replicates? How many peaks can you cann per condition? If you knockdown is good you should see notably fewer peaks in that condition. If you have replicates you can make things fancy via pairwise comparisons with something like
edgeR, but I would anticipate this is not going to be necessary. Maybe a simple browser screenshot of several representative regions might be enough, depending on how many peaks you can call.@AT point Thank you , i got my answers