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ChIP-Sequencing Tips for Small Samples

April 28, 2010 ChIP sequencing (ChIP-Seq) has revolutionized the way researchers can study protein-DNA interactions, enabling genome wide protein binding profiling in one experiment, rather than looking one locus at a time. Not satisfied with pushing just one envelope, today’s researchers are now moving towards samples that are very limited. Fortunately, ChIP methods have aged finer than a Burgundy wine so it’s now much easier to get great data using minimal sample.

ChIP Seqing and Finding in Small Samples

So if you’re ready to shelve the standard old cell lines and heterogeneous tissue samples to check out the action in primary cell lines, biopsies, or stem cell cultures, check out these recommendations from the SOLiD Sequencing scientists over at Life Technologies who were gracious enough to provide some of their key ChIP-Seq tips. Unlike those from your financial planner, these tips will set you on the path to nabbing big returns with minimal input by focusing on these three key areas:

Chromatin Shearing: Feel the Buzz

After you’ve got your hands on the isolated chromatin from your samples, the first critical step in the ChIP-Seq process is shearing it into little bite-sized fragments. This can be done in two ways:

• Enzymatic Digestion: These approaches use restriction enzymes to cleave DNA into smaller fragments. This method is nice and simple, but it can be challenging to optimize fragment sizes.  There can bias issues too. “The worry is enzymatic digestion will not produce random segmentation and certain loci will then be overrepresented, providing a false sense of your biological story.” explains Loni Pickle, Scientist II with Life Technologies’ Sequencing Group. Plus, enzymatic digestion gets blocked by formaldehyde crosslinking, and without it, nucleosomes may rearrange during the digestion process, or proteins and transcription factors in DNA binding complexes could be too far away from your DNA of interest to detect.
• Sonication: Sonication uses high frequency sound waves to break up DNA and is very effective, but the instruments require careful optimization for cell-line or tissue type and desired fragment size. Sonication has become the shearing weapon of choice recommended in most protocols, probably due to all of the cool sonicating equipment out on the market allowing users the flexibility to control fragment size for the best resolution and sequencing applications.

In ChIP-Seq the fragment size is especially important in order to create good sequencing libraries. In standard ChIP assays you might shoot for fragment lengths anywhere from 200-1000 bp, but for ChIP-Seq it’s a bit shorter, 100-300 bp on the SOLiD platform for example. Pickle suggests optimizing your shearing conditions ahead of time before diving into an experiment. “We recommend doing up front verification on your cells of interest by running treated chromatin lysates on a 1.5–2.0% agarose gel with a 100-bp ladder to determine fragment length.”

Chromatin Shearing Quality Control Tips:

• Run your chromatin out on an agarose gel to double-check fragment length and confirm that your shearing protocol is dialed in.
• If the recommended settings on your sonicator aren’t quite doing the trick, try adding additional cycles rather than increasing power settings which could bust up the complexes you want to look at.

ChIP Antibodies: Choose Wisely

No matter how well designed the rest of a ChIP-Seq experiment is, the results will only be as good as the antibody you choose, so make sure to find a good one.  “Whenever possible, use an antibody that is qualified for ChIP,” says Pickle. Many antibody providers indicate which of their antibodies are ChIP grade antibodies, meaning they’ve been tested in the ChIP application, not some other standard QC like a Western. Other suppliers might even have a separate list like this one from Invitrogen: Invitrogen ChIP Antibodies.

“If ChIP grade isn’t an option, an antibody going into ChIP first must be confirmed as specific and well characterized.  Once specificity is confirmed (for instance by dot-blot), then those antibodies recognizing native protein conformations (i.e. tested in immunoprecipitation) may have the best chance to work well in ChIP. ” advises Pickle.

ChIP grade or not, any new antibodies will need to be tested before you can trust them in an actual experiment. Also, don’t forget to include proper controls that you know are working when testing a ChIP antibody such as H3-K9Me3 at Sat-2, H3-K9.Ac at c-Fos, and negative control antibodies like Rabbit IgG and Mouse IgG for comparison.

ChIP Antibody Quality Control Tips:

• Do your best to find a "ChIP-Grade" antibody from a solid supplier. Don't waste your time with unreputuable vendors.
• Validate your new ChIP antibodies with each cell-line or tissue you want to test in a reliable assay like PCR, and make sure to use positive and negative controls for comparison.

ChIP DNA Purification: Cleanliness is a Virtue

Using fewer input cells to prepare a sequencing library makes background binding a big issue. Meaning you’ll want to find ways to get your IP’d sample as clean as you can while losing as little of it as possible along the way.  Here are a few things to think about that will help you get DNA that’s ready to roll into a sequencing library.

• Ditch the columns and phenol:Avoid carry-over of toxic phenol/chloroform, and other reagents like ethanol, EDTA or Proteinase K, that can interfere with downstream library prep reactions or qPCR. Switching from phenol or columns to magnetic beads lets you use a multi-channel pipette, makes your DNA easier to wash with better recovery and means you can stop playing that game of “Find the DNA pellet”.
• Wash, Wash, Wash: Wash steps are key to removing contaminants. Magnetic beads have made wash steps painless and more efficient. As long as you’re careful not to lose that sample, you can never wash enough.
• Knock off the blocking reagents: Carrier DNA is sometimes used to reduce ChIP background or purify small amounts of ChIP DNA, but that can wreak havoc during sequencing by getting co-purified along with your sample DNA and causing headaches in your analysis.

ChIP DNA Purification Control Tips:

• Use magnetic beads
• Wash samples thoroughly
• Avoid blocking reagents

ChIP DNA Quality Control

Now that you’ve been ultra-diligent in preparing your samples, you’ll want to take a minute to perform a few final quality checks before diving into the more time-intensive areas like library construction.

Make sure you use a control DNA sample to measure differential enrichment. Prepare a reference control library using the Input Control (the non-immunoprecipitated control sample). Yeah, we know it sounds obvious, but you’d be surprised how many people skimp on sufficient controls.

Finally, there’s no replacement for a functional QC. Pickle recommends performing PCR on your samples to check for amplification efficiency and the quality of your DNA. “Before going into library prep, use proper negative IgG and positive (for example H3-K9Me3 at SAT-2 and H3-K9Ac at c-Fos) control antibodies to assess specific enrichment of ChIP by qPCR.  Not every sample needs to go into library preparation for sequencing, but enough samples need to be ChIP-qPCR validated in the experiment to show a convincing story (that everything worked.)…and don’t forget the fragments are now 100-300bp so some of your qPCR primers may not work!  Adjust and check the amplicon size before running qPCR”, Pickle advises.

Keep these tips in mind and you’ll be pumping out killer ChIP-Seq data in no time, and be the envy of your lab-mates!

Thanks to Life Tech’s R&D team, especially Loni Pickle, for all of the inside tricks of the trade.

Check out those tips put into action in the Invitrogen MAGnify™ ChIP Kit.