Adaptors for MeDIP-seq

[nouba]

Hi!
I am all new here, so please have some patience with my stupid questions. I have looked everyone and not found a response, I'm getting so frustrated!!
So to my question: What is the difference between methylated and non-methylated adaptors (apart from the obvious fact that they are methylated?) What difference does it make for sequencing?
Furhermore, I would like to know what kit and adaptors to use for MeDIP-seq? Apparently adaptors from Illumina are methylated and I cannot use them?? OR have I misunderstod everythin? pleaseee help me!

[hduborjal]

Hi,
Methylated adpators are just protected from Bisulfite conversion. By default Illumina's adaptors from low throughput kit are methylated. There is no negative effect on the library. It can be use dfro any kind of lib.

[nouba]

Hi thank you for your reply.
But this I have understood reading online, I am still confused on the molecular mechanisms behind the difference between methylated and nonmethylated adaptors. For eg, Why would you need to use methylated adapters from bisulfite sequcening if they are protected from methylation?

[hduborjal]

Methylated adaptor are protected from bisulflite treament which converts unprotected cytosine to thymine.
If you want to carry out a WGBS project, you can produce library with methylated adpator. Then you treat the lib with bisulfite. As the adaptor are methylated, the cytosine are conserved and you can amplify the library by PCR and by using the classical PCR oligo.

[Genohub]

When to use methylated and non-methylated adapters

You need to use methylated adapters for Bisulfite-Seq and non-methylated adapters for MeDIP. Methylated adapters are needed so the cytosines are not converted during bisulfite conversion. For most MeDIP applications you'll need non-methylated adapters, because pull down occurs after ligation (and you don't want adapters being pulled down during methyl enrichment).

- Genohub

[Bioo Scientific]

Hi Nouba,

Bioo Scientific offers the NEXTflex Methyl-Seq Kit which is designed to simplify MeDip-seq and MeCAP-seq. Additionally we offer sets of up to 192 unmethylated barcodes for multiplexing. Let me know if you would like me to send you a manual for the kit.