Chromatin Shearing
Chromatin Immunoprecipitation (ChIP) has become a powerful tool for evaluating in vivo interaction of proteins with specific genomic DNA regions and plays a crucial role in helping to better understand the mechanisms of gene regulation, DNA replication, and DNA repair.
The process of ChIP involves fixative treatment of live cells with formaldehyde generating a chemical cross linking of DNA bound proteins. The cells are then lysed, and the chromatin is sheared mechanically or enzymatically, in order to reduce fragment size and increase resolution. The resultant sheared complexes are then immunoprecipitated with antibodies specific to the protein of interest, and the DNA fragments analyzed for specificity using real time PCR, sequencing, or microarray hybridization.
Until now, despite the obvious limitations, bath and probe type sonicators -- and enzymatic fragmentation using the sequence specific micrococcal nuclease -- have been the techniques of choice for shearing chromatin. With the increased sensitivity of certain post ChIP applications, researchers' need of a gentle yet more efficient method of chromatin shearing has increased.
Probe sonicators, and bath sonicators such as Bioruptor™, which operate at 20kHz range, expose the entire water bath to sonication waves and cavitation, and generate quite a bit of heat in the sample being processed, potentially denaturing proteins and stripping the DNA/protein complexes. This adversely affects the quality, quantity, and specificity of subsequent immunoprecipitation.
The Covaris S2 instrument, on the other hand, utilizes a 0.5MHz concave solid state transducer. The shorter wavelength allows for focus and more precise energy control to the sample, enabling isothermal, non-contact processing of samples. It directs the cavitation induced shearing forces only inside the samples tube. This focused and controlled isothermal method of processing not only can generate narrower distribution of sheared fragments, and generate highly reproducible shearing results, it is also less likely to strip protein-DNA complexes. In addition, the non-contact, closed-vessel processing gets rid of the worry for any possibility of cross-contamination.
Methods:
Two independent ChIP-Seq experiments were carried out on a breast cancer cell line by Dr. Seongho Ryu at Cornell University. Subsequent to formaldehyde fixation, and nuclei preparation, 2x107 cells were sheared using a Covaris S2 instrument in a 1ml volume. A time course shearing experiment ranging from 5 minutes to 25 minutes was carried out to determine the appropriate treatment time required for that specific cell line and cell density (Figure 1). The 25 minute shearing sample was chosen for further processing since it generated a shearing size range of 200-700bp.
The sheared sample was immunoprecipitated, reverse cross linked, and proteinase K treated. The DNA was then phenol/chloroform extracted, and an aliquot run on an agarose gel (Figure 2). The remainder of the purified DNA was used to generate a sequencing library for use on the Illumina Genome Analyzer.
Click the link below for detailed protocol:
Chromatin shearing with non-ionic detergent buffers. If you are using an SDS containing buffer in your chromatin shearing protocol, please contact Covaris
Results and Discussion:
Chromatin shearing using Covaris technology delivers the desirable 200-700bp fragments (Figure 1 and Figure 2). In addition, sequencing results and analysis of the two independent Covaris sheared ChIP experiments showed that 38% of the sequencing reads mapped to the genome (Table 1). The correlation of the independent experiments was quite high with an r2 of 0.94 (Figure 3) indicating a high goodness of fit for the two independent experiments. This clearly exemplifies the reproducibility of the non-contact isothermal controlled chromatin shearing using the Covaris AFA technology.
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Figure 1
Shearing Time course run: 0, 5, 10, 15, 20, and 25 minutes of Covaris processing, using a treatment condition of 20% Duty Cycle, 8 Intensity, 200 Cycles per burst, 60 seconds per cycle
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Figure 2
Gel of DNA fragments after Immunoprecipitation enrichment (samples selected for enrichment are after 20-min Covaris treatment, among the two time course runs shown above).
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Table 1
Statistical summary of sequencing results— high percentage of mapped reads for the two duplicate experiments.
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Figure 3
The close correlation of the two duplicate experiments demonstrated good reproducibility of the Covaris technology.
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