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  • ChIP-Seq mouse embryonic sample prep

    I am planning for to do a ChIP-seq with chromatin prepared from embryonic sample(E7.5 mouse embryo). For fixing the protein-DNA complex I have used 1% Formaldehyde @ 37 C for 10min.
    Sonication cycle= 30 seconds ON and 30 seconds
    No of cycles= 60
    But still after such high number of cycles I am unable to get the desired fragment size of 200-400bp for ChIP seq.
    What could be the possible modification I could do to improve the current situation?

  • #2
    ES cells are much more difficult to crosslink and sonicate properly. Their chromatin is much more compact. You first need to optimize this. Start by varying the time and temperature of the crosslinking. 10 min at 37C is obviously way too much.

    If formaldehyde ends up being too strong of a crosslinker for your ChIP target you may need to first use EGS or DSG then finish with a low concentration of FA. There are a few protocols that have been published using these alterantive crosslinkers.

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    • #3
      If you can not get this to work Active Motif has a ChIP-Seq Service that does the whole project. We have done lots of ES ChIP-Seq.

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