Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • ChiP-seq - ThermoScientific Pierce Kit - possibly overfixed DNA

    Hi, newbie to ChiP-seq. I have two libraries prepared with the Thermo Scientific Pierce Magnetic ChIP kit, micrococcal nuclease digestion and sonication. QC from an Agilent Tapestation shows several low peaks at ~180bp, 363, 537 and 713bp respectively. Normal practice I'm told is to pick at 180bp and generate libraries from that. With such a tiny peak there, I could either:
    - stick with 180bp, but not have much material to generate my library from
    - pick from several peaks but then end up with fragments of different sizes, which could make the bioinformatics hard.

    So would I:
    - do the size selection and end up with a small amount of material
    - skip the size selection (or rather have a broad size selection) and deal with the consequences later

    Any advice appreciated, this experiment is unlikely to be repeated so I'll need to work with what I've got.
    thanks.
    Last edited by Elsie; 05-18-2014, 07:49 PM. Reason: Clarify question.

  • #2
    If you prepare libraries with DNA samples described there, there will be two downstream issues. Firstly, accurate quantification of libraries for clustering with multiple peaks is difficult and inaccurate quantification can result in over or under clustering which will affect read number or quality or both, although that might be considered sequencing service provider problem. Secondly, small fragments are more efficient in cluster formation in comparison with large fragments and more of your reads will be from sequencing smaller fragments.

    Comment


    • #3
      Thanks Nucacidhunter, your comments are exactly what I suspected would happen downstream. Unfortunately these libraries are not going to be remade so I will just have to work with what I've got. Thank you.

      Comment


      • #4
        One suggestion I have reluctantly because I have not tried it myself, is shearing pool of your DNA with a Covaris set to 150-200 bp if you have access to one. In this case smaller fragment would not shear any further but larger fragments should shear. You can try shearing a cheap source of DNA to your current peaks. Pooling them to emulate your current ChIP DNA pool and trying shearing on them first to ensure that it works and that also will allow you to optimize if things does not work out at first go.

        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
        10 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, Yesterday, 06:07 PM
        0 responses
        9 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