Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • negative control or not

    hi, everyone, I am a new one in this area.
    now I plan to use this method to study a TF binding sites.
    I prepare two biological replicates, but whether I need a negative control sequencing?
    I read some paper, and say there are two case can choose:
    one-sample analysis where only a ChIP'd sample is sequenced
    two-sample analysis, where both a ChIP'd sample and a negative control sample
    I was a little confused and want to hear your opinion.
    thanks very much

  • #2
    You should definitely have a negative control for ChIP-seq.

    Comment


    • #3
      Do the negative control (normal rabbit serum), if your ChIP goes well the negative control will not have enough DNA to sequence as determined with QuanIT HS (QubeIT) DNA assay kit.

      You absolutely have to sequence your input DNA alongside your ChIP'ed DNA. That sets your baseline.
      --------------
      Ethan

      Comment


      • #4
        thanks to both of you
        I have more questions.
        You known, I am working on a rice transcription factor, in order to do the ChIP-seq, I fusion the Flag tag with my TF and transformed into the mutant background. So I have two choice about the negative control:
        1.mutant sample with Flag anti
        2.transgenic sample with no anti
        which one is better?
        by the way, how many biological replicates should I seq in my anti sample? two is enough?

        Comment


        • #5
          provided you get enough/comparable amounts of DNA pulled down i would prefer option 1 (mutant sample with anti-flag).

          just to get it right: your intention is 'just' to map transcription factor binding sites?
          if yes, i would simply sequence the input (and not the mutant sample with flag anti). reason: you can use the input to control any other ip with different antibody. the mutant sample with anti flag is only good for comparison with your flagIP.

          what do you define as biological replicate? also different transgenic lines? will the expression of your construct vary between replicates? if yes, replication is a good idea. if no, i would not replicate in the first place. one sample, have a look at the result, check some loci with qpcr compare with literature.. and maybe do a replicate later. often a single expt is enough for most of the mapping.
          maybe you also should consider pooling of replicates before sequencing if there are strong variations expected.

          Comment


          • #6
            got it, many thanks of mudshark
            all of your answer were helpful

            Comment


            • #7
              I assume you are using a FLAG-tagged approach because you do not have an antibody to your protein of interest. If that is the case then I strongly recommend you produce your own antibody. It is a easy and simple thing to do and the cost in terms of labor and money is minimal in terms of the cost of the ChIP-seq project. Yes it takes 90 days, but too often I see people two years into a project and still trying to get by with no or a low quality antibody. How to make good antibodies is a whole different topic so I'll leave it at that.

              As far as your last question, I don't understand what you mean by 'mutant'.
              --------------
              Ethan

              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
              11 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, Yesterday, 06:07 PM
              0 responses
              10 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 03-22-2024, 10:03 AM
              0 responses
              51 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 03-21-2024, 07:32 AM
              0 responses
              68 views
              0 likes
              Last Post seqadmin  
              Working...
              X