Hi,

MeDIP protocol works with single stranded DNA. So the first step is to denature DNA.

In this case, it's better to ligate adaptors before on double stranded DNA and then perform the IP. Just take care that the adatpors are not methylated.

However, if you want to ligate after IP, you have first to repair DNA and get back to dsDNA. But it can introduce potential bias.

We have a protocol for MeDIP-seq here below. Don't hesitate if you need more info

https://www.diagenode.com/protocols/medip-sequencing-protocol

Hi

After you shear the DNA, size select them , end repair and add 3'A, do the adaptor ligation. next is the MeDIP.

Hi,

Yes it is. once you have your adapter ligated, you can start the standard MeDIP protocol with denaturating the DNA. Then IP, washes and final elution. After that, you will perform the amplification of your libraries.

Do let me know if you have any other question or if you want to be contacted by someone local.

Hi, thanks for answers! I have successfully done the MeDIP using NEBNext Ultra II adapter ligation prior to denaturation and immunoprecipitation. After elution, I continued the kit protocol with the size selection and amplification followed by PCR amplification.

Hi,

I would like to ask two questions related with this topic.

1. Why it is necessary to denature the DNA prior to IP?

2. If you ligate the adaptors after IP, why can it introduce potential bias?

Thanks

Hi,

5-mc antibody only recognize methylation on single stranded DNA.

https://www.diagenode.com/en/p/5-mc-monoclonal-antibody-33d3-premium-100-ug-50-ul

That's why it's not possible to ligate your adapter after IP because you end up with single stranded DNA. So if you really want to do that, you have first to repair DNA and recover double stranded. And this step can potentially introduce bias.

https://www.diagenode.com/en/protocols/medip-sequencing-protocol