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MeDIP-seq protocol with TruSeq adapters

MeDIP-seq protocol with TruSeq adapters

10 Comments
  1. Silvia permalink

    Hi! I’m trying to perform Medip-seq. First of all, I ordered unmethylated oligos to construct my own TruSeq adapters. When I performed a library preparation with Illumina TruSeq DNA kit, my home-made adapters made a lot of dimers compared with original Illumina adapters and the ligation efficiency was very very low. Do you perhaps have any special tips on adapters preparation? I suspect that the oligo with the final T was not very good and maybe this could be the reason why I observe a lot of adapter dimers. Thank you!
    Silvia

  2. ethanomics permalink

    Hi Silvia,
    I am a little too busy to answer with much depth, but my guess is that you did not do 4 cycles of PCR before size selection as I recommend in my protocols. You need to convert the Y-shaped adapters to dsDNA before size selection. I have some blog posts concerning ChIP-seq. I am getting really good data using the protocol I have posted here. I think I have a slightly updated version but I’ll try to post it in the next week but what is up is good. On the bioinformatics side I am finding MeDIP-seq data a little hard to work with…..I could go on and on about this. But on the wet lab side things seem to be working great.

    Ethan

  3. Roger permalink

    Hi there,
    I have a question related to your protocol. What is the benefit for using the MeDIP-seq blocking oligo in step 98? Do you add this always or only when you use the methylated adaptors from illumina? Thanks

  4. ethanomics permalink

    I use the blocking oligo to keep the fragments from annealing by their adapter sequences. The published MeDIP-seq protocol doesn’t use this so it works without. But I imagine it reduces background, but I haven’t tested that. I am actually surprised it works without the blocking oligo.

    Ethan

  5. Frustrated PhD student permalink

    Hi Ethan, Thank you for a very good and informative blog! Especially to persons like me who are so new to this field!
    My question is very basic. What is the difference between methylated and non-methylated adaptors on a functional and molecular level? (apart from the obvious fact that they are methylated?) I know that the methylated adaptors are only used for bisulfite sequencing (somehow they are protected from methylation for this method??) and that for MeDIP-seq I cannot use methylated adaptors. What difference does it make for sequencing? What is the reason?

    Also I saw that you mentioned that all adaptors from Illumina are now methylated? So what should adaptors should I use for my MeDIP-seq (if we are going to run on Illumina HiSeq).
    I hope you will see this and can help me!!! Thanks again for a great blog!

  6. ethanomics permalink

    Hi Frustrated PhD student, the only reason to use methylated adapters is for bisulfite sequencing. I don’t know why Illumina included methylated adapters in a lot of their kits especially when it is not something that made clear which kits include them and which do not. I think they are moving away from methylated adapters as they are expensive to synthesize. You should definitely contact Illumina it you are wondering if the kit you have has the adapters methylated or not. Or better just order them from IDT. I’d bet that’s where they get them from.

    Ethan

    • Frustrated PhD student permalink

      Thank you for the response! And again thanks for a great blog!

  7. Hervé permalink

    Hi Frustrated PhD Student.
    Like Ethan, we also had the same problems using methylated adapters from Illumina for MeDIP-seq.
    Finally we are now using a kit from Bioo Scientific that works well and specifically designed for MeDIP-seq. Their adaptaters are not methylated and compatible with Illumina HiSeq.

    Hervé

  8. Frustrated PhD student permalink

    Hi Hervé!

    Thank you for the info, good to know that I am not alone! What is the kit number from bioscientific? I am for the moment using bioscientifics kit for ChIP-seq library preps and I am very satiesfied!

  9. ethanomics permalink

    The ChIP-seq kit is good because they copied the protocol from me, or at least they came out with it a few months after I started posting my protocol around the web and it is exactly the same protocol. Thumbs up to their R+D department. As far as the adapters, I’d just order them from IDT. I’m nearly certain that’s what Bioo does.

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