I've performed ChIP qPCR on my samples using positive control regions and determined the enrichment relative to negative control regions using the delta delta Ct method. I'm seeing enrichment in of ~2-4-fold depending on the IP (H3K4Me1 and Me3, H3K27Ac and ER). I had to include two washes each using the RIPA 500 and RIPA LiCl buffers on my Protein A/G Magna ChIP beads (Millipore) to see this enrichment in the first place. Should I optimise the washes further (and if so how many more should I do?) before sequencing or is this sort of enrichment likely to be good enough to produce good ChIP-seq data?
Thanks!
What kind of antibody are you using prior to IP the samples? Is there any degree of inespecific IP? I think you should get better results if you optimize the amount of antibody and also the conditions of imunoprecipitation. If you do not have good IP I believe that further washes are meaningless.
Good luck and all the best.
I've already seen that increasing the number of washes (as described above) has lead to some enrichment so I think it is reasonable to assume that further washing could increase the enrichment. How many washes do people do?
I'm using well validated antibodies for the ChIP so the problem is unlikely to lie there.
There is ~2-fold enrichment from the IgG control as measured against the genomic input using a negative control region but my H3K4Me3 ChIP shows a further ~2-fold enrichment over the IgG control. I feel like I should be seeing better enrichment before I proceed to ChIP-seq and I am wondering what other people's experience is?
Hi Dylan,
If you have around 2-fold enrichment in the IgG control I agree with you that you should optimize the protocol a bit more. As you previously noted, washing more is always a good idea, and the limit of additional washes that can be done depends on the amount of recovered DNA you get in the end. In other words, wash as much as you can as soon as you get enough DNA to prepare the library. You mentioned you did two washes and you could definitively increase that: in a standard ChIP protocol you first wash with a low salt buffer, then a high salt, followed by LiCl buffer and finally TE buffer.
Hope that helps, good luck!
Antonio.
Hello,
Or you can use validated kits: https://www.diagenode.com/p/ideal-chip-seq-for-histones-library-preparation-package-incl-index-primer-set-1-24-rxns :)
I agree with Dylan. When I do the wash steps, I always following the standard steps: IP buffer for twice, then low salt buffer and high salt buffer for one time. After that, wash the pellet once with TE buffer and change the tube with a new one. Finally, wash the pellet with TE, and then do the reverse cross link process at RT. By the way, I think the fixation and IP process are also critical for got enough DNA. In addition, more original tissue will assurance the amount of DNA.
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