Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • MeDIP-seq - input sample needed?

    I've prepared some MeDIP-seq libraries and they appear pretty good and ready to be run on the HiSeq. My question is, do I need to run input control libraries like we do with ChIP-seq or is the fragmentation of the genome and subsequent amplification so even that it is not necessary?

    I have samples from several cell lines and was thinking of just running one input control?
    --------------
    Ethan

  • #2
    Input controls are not a bad idea, what did you use to fragment?
    Are you using methylated or non-methylated adapters?

    Comment


    • #3
      I used the Covaris S2 and their suggested settings using microTubes.

      For adapters, I used adapters with the TruSeq sequences synthesized by IDT and non-methylated. I am aware that the TruSeq adapters from Illumina are methylated.

      My libraries have about a 50-fold difference in signal between what should be a methylated region and a CpG-less sequence using qPCR and normalized to input. From what I understand this is good enrichment for MeDIP. Do others see better enrichment?
      --------------
      Ethan

      Comment


      • #4
        that will work. we sequence material with 40-60 fold greater signal. let me know how it turns out.

        Comment


        • #5
          Originally posted by ETHANol View Post
          My libraries have about a 50-fold difference in signal between what should be a methylated region and a CpG-less sequence using qPCR and normalized to input. From what I understand this is good enrichment for MeDIP. Do others see better enrichment?
          hey,
          I always compare methylated regions to unmethylated regions of similar CpG density to measure enrichments - that way you avoid getting into sonication artifacts. Especially if you are testing your libraries and not the IP material for enrichment.
          E.g. use a methylated CpG island vs an unmethylated one (take for example a housekeeping gene promoter). Don't know what cell type you're using but methylated CG islands are mostly at sperm specific promoters or imprinting control regions. Even if only one allele is methylated, it will still give you a good signal to measure enrichment. Stay away from repeats and transposons.

          Comment

          Latest Articles

          Collapse

          • seqadmin
            Strategies for Sequencing Challenging Samples
            by seqadmin


            Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
            03-22-2024, 06:39 AM
          • seqadmin
            Techniques and Challenges in Conservation Genomics
            by seqadmin



            The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

            Avian Conservation
            Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
            03-08-2024, 10:41 AM

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by seqadmin, Yesterday, 06:37 PM
          0 responses
          8 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, Yesterday, 06:07 PM
          0 responses
          8 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          49 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          67 views
          0 likes
          Last Post seqadmin  
          Working...
          X