ChIP-Seq has transformed the field of epigenetics, providing unprecedented resolution and coverage for surveying protein- DNA interactions. Powerful, high-throughput sequencing is credited with most of the benefits, but there’s a lot more that goes into ChIP-Seq than sequencing. Chromatin shearing is one of the first critical steps in the ChIP-Seq workflow. Depending on your approach, you will either faithfully represent the biological scenario you wanted to study, or disrupt epitopes and introduce all sorts of bias so you won’t actually know what’s relevant downstream.
Chromatin Shearing Techniques
As with any scientific approach, there’s multiple ways to get from A to B. Chromatin shearing is no different. Two general approaches are still widely used today: enzymatic digestion and mechanical shearing. Despite sharing a similar end goal, these methods couldn’t be more different in their approach; one uses a naturally occurring enzyme, micrococcal nuclease (MNase), to digest DNA, while the other uses acoustic energy to shear DNA into smaller fragments. Let’s take a closer look at each of these.
In this guide…
- Popular chromatin shearing approaches
- Discuss when you might opt for one vs. another
- Share considerations for other aspects upstream that can impact
For years, micrococcal nuclease (MNase) digestion of chromatin has helped researchers in nucleosome mapping experiments, but it’s utility doesn’t necessarily end there. This enzyme, which generally cuts linker DNA connecting between nucleosomes can be used in other chromatin-related analyses as well.
Advantages of MNase Digestion
Researchers often opt to use MNase digestion in their ChIP experiments for a number of reasons.
- It doesn’t require any expensive equipment
- It can create high resolution maps since it generally digests to the ends of nucleosome-bound DNA
- The reaction is isothermal and can be milder than sonication approaches, resulting in minimal damage to epitopes of interest
- It is essential when working with native, or non-crosslinked chromatin (e.g. histone modification studies) where sonication would disrupt protein-DNA complexes
Drawbacks of MNase Digestion
Despite its’ advantages, enzymatic digestion is far from perfect. There are a number of limitations that may be more or less meaningful depending on your experience level and experimental scenario.
- MNase does exhibit sequence specific cutting, so there’s an opportunity for bias
- Nuclear accessibility to MNase can vary in cell types and model systems, so optimization is often required
- Different preparations (lots) of enzyme can have varying levels of activity, so it is often necessary to qualify before using
Whenever you’re using an enzyme as a tool, there’s going to be variables to consider and adjust. MNase digestion is no different. Here’s what some R&D leads familiar with the method had to share: Michael Sturges, Sr. Product Manager at EMD Millipore expands, “Reaction conditions will vary depending upon amount of material (cell equivalents), concentration of enzyme, and time of digestion. Undigested material is often spun out of the sample following digestion since this is largely insoluble.”
“As with any enzymatic process, temperature, sample concentration, and salt content can greatly affect the reproducibility and robustness of the process. These parameters need to be tightly controlled with each sample to assure the shearing is reproducible.” J.D. Herlihy, Product Manager at Covaris
Mechanical Shearing of Chromatin
If enzymatic digestion isn’t a good fit given your experimental situation, or experience, then fear not; there are number of different derivatives of mechanical shearing using acoustical energy to choose from. Chromatin Sonication Ultrasonic energy has been used in the lab for years to disrupt everything from cell membranes to chromatin. Unlike, enzymatic digestion, sonication uses energy to shear chromatin into smaller fragments. In addition to wearing some stylish earphones, sonication brings several advantages to the table for chromatin shearing.
Advantages of Chromatin Sonication
Unlike MNase digestion, there are no enzymes involved here. Generally that means these approaches will be less susceptible to sample-to-sample variation and might see less variation in different types of species and tissues. Also, since sonication is mechanical in nature, there are no issues with sequence bias etc., that can sometimes be problematic with MNase digestion.
Dr. Kyle Hondorp, R&D Scientist at Active Motif shares more on this, “Sonication will perform better in difficult to lyse cells such as T cells. If complete lysis does not occur the enzymes may not gain access to the nucleus, thus resulting in incomplete digestion. Rigorous sonication will not only shear DNA but will also aid in the lysis of difficult to lyse cells, thus improving yields.”
Another general benefit of sonication is that it is often easier to obtain a more homogenous sample population that can be “fine-tuned” to a certain length. This makes it useful when using different types of downstream applications, where you may desire a longer or shorter fragment size. There are a number of types of sonication platforms in use today including direct probe sonicators, Cup Horn sonicators, and focused ultrasonicators to name a few. Let’s take a closer look at each of these to see how they compare.
Direct Probe Sonicators
As the name suggests, these sonicators use a probe immersed directly into a sample tube. Probe sonicators have been used in the lab for years and are definitely useful. They deliver a whole lot of power to the sample, which makes for short sonication times.
That said, this isn’t always ideal for ChIP applications. First of all, since you’re using a probe that interacts directly with a sample, there’s always a risk for cross contamination when sonicating multiple samples, which is already a bit laborious since you can only sonicate one sample at a time.
Additionally, the energy can agitate samples to the point where they foam. This can cause headaches, particularly when you’re working with smaller volumes. Lastly, the heat form the direct energy can also introduce another variable to optimize as it can also degrade samples further.
If your lab is well into its ChIP-based studies, it might be time to move towards other options though that are better suited to accommodate more throughput.
Cup Horn Sonicators
Instead of applying direct energy to samples, Cup Horn sonicators use a separate water bath to indirectly deliver energy to your sample. Since this type of a water bath approach is less intense than direct sonication, the duration of sonication required can be a bit longer.
Unlike probe sonicators, Cup Horn sonicators can generally process multiple samples simultaneously and since samples remain in a sealed tube, there is minimal risk of cross contamination. Cup Horn sonicators generally require more energy as they are energizing the entire water bath in which the samples sit. This can sometimes lead to unwanted heat, so having a cooling mechanism in place will help control unwanted degradation.
Acoustic systems can differ by more than the probe/sample interaction. There are also different levels of frequency and corresponding wavelength of energy used to shear chromatin. Ultrasonication platforms such as Covaris’ Adaptive Focused Acoustics™ (AFA)-powered platforms use a higher frequency (500kHz) when compared to other sonicators (20kHz).
This higher frequency energy results in a smaller wavelength applied to the chromatin samples. Why should you care? Generally, the smaller the wavelength, the more precisely it can be targeted so less power needs to be applied to do the job.
Less power means less heat beating up the protein-DNA interactions you may want to map. Covaris’ J.D. Herlihy explains, “Other mechanical shearing methods (sonication) generate heat during processing that is ineffectively dealt with by ice baths or cooling mechanisms. The excess heat damages proteins, DNA, and formaldehyde cross-links; decreasing sensitivity, reproducibility, and reliability of the assay.”
The latest entry into the ultrasonicator class is the Active Motif PIXUL Multi-Sample Sonicator, which was designed to address the limitations of previous multi-sample sonication technologies. PIXUL uses an array of multiple transducers and lenses to focus ultrasonic energy to each sample well, allowing it to sonicate up to 96 samples at the same time using common and inexpensive 96-well cell culture plates.
The power of PIXUL was demonstrated in a recent Nucleic Acids Research article that compared it head-to-head with the competition, and found that it more than held its own. According to Active Motif Product Manager Dana Meents, “PIXUL is perfect for simultaneous sonication of multiple sample types within a single run, as well as the optimization of sonication conditions for new sample types since up to 12 different sonication programs can be used in each run. Based on that, we believe that the PIXUL sonication platform really is the best instrument available for high-throughput chromatin shearing.”
The Shearing Breakdown
With chromatin shearing, there are many options and approaches. The benefits of each of these will vary depending on your sample, model, experience, and practical considerations like budget and available lab equipment. Most of these approaches will require some level of optimization and tweaking.