Chromatin Analysis Support—Getting Started

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. 

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.