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Chromatin Analysis

For each ChIP reaction, the MAGnify™ Chromatin Immunoprecipitation (ChIP) System recommends using 10,000-300,000 cells or 0.167–5 mg of tissue. The Thermo Scientific™ Agarose ChIP Kit and Thermo Scientific™ Magnetic ChIP Kit recommend 2–4 × 106 cells per ChIP reaction. To ensure consistency and decrease experimental variability, we recommend preparing a common chromatin batch suitable for multiple ChIP experiments. 

Many factors influence yield, including cell type, target protein, antibody, etc. Generally speaking, anywhere between 0.5 ng to >10 ng can be obtained per ChIP reaction. Yields are usually too low to be measured using a NanoDrop™ spectrophotometer. 

Both sonication and enzyme digestion are widely used for ChIP. Micrococcal nuclease results in greater reproducibility and allows better control of the fragment sizes. However, it can take more optimization and the enzyme can be compromised and fail to work properly. 

We recommend the following controls:

  • Negative control antibody: Either do not use a primary antibody, or use the normal rabbit IgG or mouse IgG isotype control. 
  • Positive control antibody: This control ensures that each step of the procedure is working. For example, we observe consistent enrichment of heterochromatin markers such as H3-K9Me3 at the satellite repeat locus (SAT-2). 
  • Negative control PCR primer: This control is designed against a sequence that would not be enriched by your chromatin IP procedure. 
  • Input DNA control: Input DNA is DNA obtained from chromatin that has not been immunoprecipitated and has been reversed crosslinked similar to your samples. It is a control for PCR effectiveness and utilized in ChIP sequencing data analysis. 

Yes. The MAGnify™ Chromatin Immunoprecipitation (ChIP) System has been validated for all major next-generation sequencing platforms. Usually 1-10 ng ChIP DNA is enough for next-generation sequencing. For next-generation sequencing support, please visit our Next-Generation Sequencing Support Center.

Yes. Please refer to this paper for chromatin preparation protocol from FFPE samples: Fanelli M et al. Nature Protocol, 2011; 6:1905-1919. Our R&D has collaborated with the authors to confirm that the MAGnify™ Chromatin Immunoprecipitation (ChIP) System is compatible with this protocol. 

For fixed but not embedded samples (e.g., 10 min in 1% PFA), the standard protocol will work. No further modification is needed.

Sonication is used to break open the nucleus, allowing the digested DNA fragments to be released and extracted. In the Thermo Scientific™ Agarose ChIP Kit, a high-salt method is used to extract fragmented DNA. In principle, both methods work well for this purpose but sonication is easier. 

Shearing of the chromatin into appropriate fragments is required to ensure optimal ChIP results. Ideal shearing should yield DNA fragments between 200 and 1000 bp in length. ~200-500 bp fragments are usually used in downstream qPCR analysis and ~100-300 bp fragments are used in massive parallel DNA sequencing downstream analysis. 

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