ChIP Optimization: Chromatin Shearing in ChIP

Loni Pickle, Life Technologies R&D Scientist, reviews various approaches for chromatin shearing upstream of chromatin immunoprecipitation (ChIP).

Tissue Processing Using Magnetic Beads -- Shearing Optimization

Chromatin shearing is the step in ChIP where I receive the most amounts of questions; so let's spend a couple of minutes reviewing this key step. There are two basic methods to shear your samples during ChIP experiments: Enzymatic digestion and Sonication.

- Enzymatic Digestion
Uses enzymes to fragment sample

- Sonication
Uses sound waves to fragment sample

Enzymatic digestion, or what's used for native ChIP, which is a ChIP with no cross-linking. This approach used is restriction enzymes to cleave DNA into smaller fragments, since it doesn't use formaldehyde cross-linking it can allow greater antibiotic accessibility to very strong DNA binding factors such as histones. But, it could also produce sequence bias. Some researchers worry that enzymatic digestion will not produce random segmentation and certain [..] will then be over represented providing a false sense of your biological story. Sonication uses high frequency sound waves to shear samples; we recommend using sonication since it is less prone to bias than enzymatic digestion.

There are two main types of sonicators:

- Probe Sonicators
Which transfer ultrasonic energy directly into samples

- Water Bath Sonicators
Which transfer ultrasonic energy through water and closed tubes into your sample

We see easier results with water bath sonicators because they tend to be more consistent user to user, easier to use, and gentler on the lysates.

Key Factors that Impact Sonication Results

Sonication and the resulting fragmented DNA can be influenced by a number of factors; of course the amount of power you deliver and the number of cycles you sonicate, can dramatically impact your size distribution; but there as some other less obvious factors. Some less obvious variables that can impact your sonication are your cross-linking conditions, if you increase cross-linking for longer durations or even use a different concentration of formaldehyde you may need to sonicate for longer to compensate.

Tube type; if you change the type of tube that your sample is in; say from a 15 ml conical to a 1.5 ml tube you'll probably need to adjust your sonication conditions as well.

Volume of liquid in your tube; if you change the volume of liquid in your tube you should confirm that your sonication results are the same. Volume can introduce variations in fragment size.

Buffer composition; this one is a biggie, the type and concentration of detergents in your lysis buffer will definitely change your fragment size. So if you make any adjustments to your buffer, be sure to reoptimize your sonication parameters.

Useful tips for Optimizing Shearing

Here's some tips that should be useful for when you are optimizing your shearing:

Keep your samples cold -- keep your samples on ice or in cold water alternating cycles of on and off ice to allow your samples to cool between cycles. This will help maintain your DNA protein interactions. Sonication produces heat, that can disrupt your protein DNA interaction.

For ChIP qPCR, shoot for a fragment size between 200 to 500 bp. For ChIP Seq applications using the SOLiD™ platform shoot for a fragment size range from 100 to 300 bp. If you're using another platform, follow that vendor's recommendations. Be sure to verify your fragment size before moving into your immunoprecipitation step of the ChIP protocol. You want to aim for the least amount of sonication cycles and intensity that gives you the correct size distribution.

So I hope this quick review and these tips will help you optimize your shearing process. As we've covered here there are a lot of factors that can influence your fragment size but I think you'll find it's well worth the effort as you head into your downstream application.

For more information visit http://owl.li/cPD2x

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