Is there anyone who ever used dual-crosslinking method in ChIP experiment? I want to ask if there are any subsequent effect on the Chromatin fragmentation step, and because increase the spacer arm could fix some indirect interactions as well, would dual-crosslinking cause false positive in the final data?
I tried a lot to get enough DNA in my experiment, but failed, so I want to try dual-crosslinking to see if I can get more relevent DNA, so if you have done such experiments, please tell me about your experience!
Thank you so so much!
What I know is that dual cross-linking can improve the result (signal to noise), even for transcription factors which are directly bound to DNA. Why it is efficient sometimes, and not always beneficial, is a mistery to me. Indirect interactions are also fixed when using only formaldehyde anyway, so I would not worry too much. You just have to adjust cross-linking time and fragmentation conditions.
Good luck
Hi,
Please ahve a look at https://www.diagenode.com/files/products/reagents/ChIP_crosslink_gold_manual.pdf
Hi
In ChIP experiment, dual cross-linking can improve the result. But You will find false positive in the final data when your fragment size is bigger. 1. Ideal fragmentation size is 200-500bp (more than 85% of your sample). Standardize shearing first
2. cross-linking time keep it 10min at 37c with mixing. After quenching, give at least 2-3 washes(1st wash in RT and after that buffer and centrifuge should be in cold condition).
I think if you follow this 2step, your problem will be solved.
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