How to test ChIP cross-linking and reversal? - (Jun/13/2006 )

Hi there,
I want to make sure that my cross-linking procedure works and was wondering if there is a general way of testing and visualizing cross-linking and reversal of cross-linking?
Thanks in advance,
ackl-d

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Cross-linking is very efficient. We are only concerned about over cross-linking. Just follow exactly what is on the protocol. While reverse crosslinking tends to be hard and a minimum of 4 hrs is required. You can safely let the reverse crosslinking go overnight.

I don't know any methods that can test the adequacy of those procedures.

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But my PI asked me not to overnight. She worried that the samples would dry out if overnight. But I am not sure 4.5 hours is enough or not.

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I reverse-crosslink for 4-5 hours and usually it's enough

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If you are interested in trying it, adding a 10% suspension of chelex in water directly to your beads after the last wash step of the IP and boiling for 10 min is sufficient for both elution and reversal of crosslinking.

We have published a method using this in Nucleic Acids Research (NAR 34(1) electronic only) and in the recently released first issue of Nature Protocols (Nature Protocols 1, 179-185). This method is robust, involves MUCH less labor (we can complete the assay, from sonicated chromatin to PCR ready DNA, in about 3.5 to 5 hours for 20-24 samples) and has replaced the traditional method in our lab as well as several others.

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hi KPDE,

I am pretty interested in your fast protocol of chIP, I have checked the nature protocol, but our university has no subscription to this journal, could you please send me a pdf reprint of it. Thank you very much.

Also, I have questions about your last elution step, after boiling, you will centrifuge to get the supernatent, so what speed and temperature you use for this step? and also as for our lab's current chIP protocols, after elution and DNA precipitation, our DNA is dissovled in 20 ul TE, and the concentration is about 30-50ng/ul (I was using about 100 million cells), I am wondering what the concentration of DNA you get by your fast method in 100 ul TE (according to your NAR paper).

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I'm sending you a reprint. In answer to your questions:

1) For each centrifugation after boiling I centrifuge at ~10,000g at 4C for a minute or two. The temp and speed cool the samples off, precipitate any water vapor, and spin down any precipitated vapor to the bottom of the tube.

2) I can't tell you the amount of DNA at the end of the protocol since we've never measured it but I can tell you that there's no decrease in yield over the traditional method. In fact the yield is often better with the Fast ChIP protocol. If you are worried about the DNA concentration being too low for your particular application you could try ethanol precipitating out of the final elution. If you are just doing PCR though, I think the DNA in the final 200ul is sufficiently concentrated (especiallly if you are using 50X more cells per IP than we do).

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I got your protocol, very nice, I am going to try it right away. Thank you very much.

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Dear KPDE and qiudao

I am also looking to a fast and useful protocol for ChIP. Is it possibile for you to send me the protocol or a reprint of the paper in nature protocols as also our library to not have access to this journal?

Thank you a lot

Danilo

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I would also like to get the protocol if possible, please

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