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  • Low DNA yield in ChIP - possible workarounds?

    Hello community,
    This is my first post, but I've been a reader of this forum for a few years now, it's a great resource, so thanks to all who contribute.

    We are doing ChIP-seq for a number of sequence-specific TFs.
    DNA yields are generally acceptable (25 ng, by pico-green), except for one TF for which we get at best 1 ng per IP.

    We've tried a lot to optimize the IP conditions (e.g. amount of antibody, amount of chromatin, buffer composition, wash conditions, etc), but to no avail.

    The IP does work in the sense that in gene-specific PCR, we do see an excellent enrichment at a few known target loci.

    We've thought of performing a large number of IPs and pooling all of them together, but we are limited by the amount of antibody and chromatin we have left.

    For a situation like ours, has anyone thought of mixing their low amount of IP'ed DNA with some amount of input DNA, to reach a total amount of DNA acceptable for library preparation? My rationale is that if the IP worked really well (it seems like it did), then it will not suffer much from being "spiked" with some input DNA.

    What do you experts think? I searched this forum and pubmed/google scholar but did not find anything about the feasibility of this approach.

    Thanks in advance for your help.

    Alex

  • #2
    I would do normal library prep with the 1 ng sample (it usually works even if you cant measure it with Qbit) but if you think spiking in with other DNA will help you recover a higher % of your fragments you could try with DNA from another species.

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    • #3
      We've made ChIP libraries with less than 1 ng of ChIP DNA. We use the NuGEN Ultralow kit and increase the PCR to 18 cycles. This usually works.

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      • #4
        Thanks for your suggestions, Chipper and NexGenSeq.
        We currently do not prepare libraries ourselves, but we may just start doing that.

        Alex

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        • #5
          Apollo works too

          We have the IntegenX Apollo 324 and we regularly make libraries for our customers at that low a quantity.

          It is a service we offer if you are interested

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          • #6
            Back in the days of ChIP-chip I was using Whole Genome Amplification kits. 1ng is good enough amount for library prep for kits currently available in the market.

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            • #7
              I would not spike in with input from the same organism. This will lead to a reduction in the ChIP efficiency and low RSC values in strand cross-correlation analysis (Landt et al 2012).

              We have sequenced libraries prepared from between 1-2 ng ChIP DNA, but this often leads to low complexity libraries and high redundancy rates (> 80%). I have seen a significant inverse correlation between starting template amount and read redundancy rate. It is definitely better to pool several ChIP elutions into one sample and sequence at least 5 ng template.

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