Chromatin shearing for ChIP

with AFA™ Focused-ultrasonicators

Chromatin Immunoprecipitation (ChIP) is a powerful technique for evaluating in vivo interactions of proteins with specific regions of genomic DNA and helps to better understand the mechanisms of gene regulation, replication, and DNA repair. Typically, the technique utilizes formaldehyde to cross-link proteins to DNA in vivo. Chromatin from cross-linked cells and tissue is harvested and sheared for subsequent immunoprecipitation (IP) of the protein:DNA complex with antibodies specific to the proteins of interest, thereby specifically enriching the DNA sequences bound to this protein. The bound DNA is purified and can be measured or sequenced by one of several analytical methods. The optimal fragment size is dependent on the intended downstream analytical methods to be employed, typically 200 to 1000 base pair fragments.

While the quality and specificity of antibodies, and other aspects of the immunoprecipitation are important to the success of ChIP experiments, the quality of the sheared chromatin starting material is essential to attaining successful results. Adaptive Focused Acoustics™ (AFA) Technology is ideally suited to providing the highest quality sheared chromatin. Isothermal and controllable shearing with AFA technology overcomes the shortcomings of traditional sonication and enzymatic methods of chromatin shearing, which damage precious epitopes with excess heating, introduce shearing biases, are difficult to optimize, and do not provide reproducible results. Covaris has developed the truChIP™ Chromatin Shearing Kits with an optimized, flexible, and universal protocol for chromatin shearing with the high performance Covaris Focused-ultrasonicators. This allows the seamless inclusion of truChIP Chromatin Shearing by AFA into any ChIP Protocol.

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AFA technology increases the sensitivity of ChIP assays by preserving precious epitopes for subsequent immunoprecipitations. Unfortunately, to achieve adequate shearing forces standard probe and bath sonicators unnecessarily heat samples, and consequently require high detergent concentrations (e.g., typically 1% SDS) to achieve chromatin shearing (AFA vs. sonication). The excess heat energy and high detergent concentrations denature proteins, damage epitopes, disrupt protein:protein and protein:DNA interactions, and introduce shearing bias; elements leading to decreased IP efficiency and sensitivity of your ChIP experiments. To provide the highest sensitivity ChIP results, Covaris has developed the truChIP™ Chromatin Shearing Kits with non-ionic and low SDS (0.1%) Shearing Buffers. The truChIP™ Chromatin Shearing Kits include all the reagents optimized for chromatin shearing with the Covaris Focused-ultrasonicators to improve the sensitivity of your ChIP assay.

FIGURE 1: Increased ChIP Sensitivity with truChIP™ Chromatin Shearing

FIGURE 1: SYBR Green TaqMan results of enriched acetyl Histone H3 over a 100 kb span of the murine IgH locus. Cells crosslinked for 5 minutes and processed for 15 min. according to the truChIP Chromatin Shearing Kit Non-Ionic Shearing Buffer protocol.

FDA Researcher—“The ChIP results obtained using Covaris shearing technology (AFA™ and truChIP™ Kit) is the most sensitive and novel that I have ever seen… These are outstanding results. The results I have generated from cross linked ChIP for acetyl H3 before are usually 10-20 fold enriched. As you can see, most of these are far far above that. Additionally, it has preserved epitopes that we never saw before.” 11 June 2010

 

FIGURE 2: Protein epitope preservation while maintaining efficient shearing

FIGURE 2: Time course of Chromatin shearing following the truChIP™ protocol: Shearing with AFA™ up to 12 min has no effect on Beta Catenin of Histone H2B epitopes as demonstrated by good immunoreactivity at all time points (insert). Red-5 Min, Navy Blue-8 Min, Green-10 Min, Light Blue-12 Min.

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Focused-ultrasonicators overcome biases observed from chromatin shearing by enzymatic and standard probe or bath sonicators. All probe and bath sonicators introduce excessive heat during the mechanical shearing process in an uncontrolled and variable manner. The heat causes biased shearing at AT rich regions due to the lower annealing temperature of AT base pairing.

Enzymes possess inherent sequence biases, compounded by the effects of chromatin structure, DNA:Protein interactions, and nucleosome positioning on specific chromatin regions. For example, the common enzyme employed for enzymatic based chromatin shearing, Micrococcal Nuclease (MNase), cleaves the linker region between nucleosomes. MNase demonstrates a bias for AT rich regions (*), and cleavage efficiency can be affected by chromatin structure and nucleosome positioning. AFA™ Ultrasonic Technology is well documented with no demonstrable sequence bias, making AFA™ the standard for DNA Shearing in Next-Gen Sequencing library preparations. Enzymes are also intrinsically susceptible to pH level and buffer and substrate concentrations, negatively affecting their performance in DNA and chromatin shearing applications.

(*) Cuatrecasas, S.F., and Anfinsen, C.B. (1967) J. Biol. Chem., 244, 1541-1547.

FIGURE 3: Superior performance of Focused-ultrasonicators™ shearing compared to probe sonicator

FIGURE 3: Shearing bias of probe sonicator demonstrated by the artifactual “hot-spots” of enrichment (arrows). The chromatin shearing with the truChIP Chromatin Shearing Kits and a Focused-ultrasonicator provide a consistent, unbiased coverage.

FIGURE 4: ChIP-Seq data from Histone H2A Immunoprecipitation

FIGURE 4: The unbiased shearing with a Focused-ultrasonicator and truChIP Chromatin Shearing Kits allows proper sequence alignment and better signal to noise for your ChIP-Seq experiments.

Sequence data kindly provided by: Ethan Ford – Biomedical Research Foundation, Academy of Athens Soranou Efesiou, Greece.

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Unprecedented control over shearing size range with the programmable and controllable Focused-ultrasonicators utilizing AFA, delivers reproducible results with every experiment. The Covaris AFA process provides optimally physically sheared chromatin for your intended application, easily accommodating all NGS platforms for ChIP-Seq assays. This means you get chromatin sized precisely and better suited for your intended use.

FIGURE 5: NGS platform specific shearing length control

FIGURE 5: The unbiased shearing with a Focused-ultrasonicator and truChIP Chromatin Shearing Kits allows proper sequence alignment and better signal to noise for your ChIP-Seq experiments.

FIGURE 6: Reproducible size specific chromatin shearing

FIGURE 6: Bioanalyzer results demonstrating reproducible chromatin shearing from duplicates samples obtained with the truChIP™ Chromatin Shearing Kits and a Covaris Focused-ultrasonicator.

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The optimized truChIP™ Chromatin Shearing Kits and included protocols were developed to work reliably with all mammalian cell and tissue types. There is a plethora of kits, reagents, and “home-brew” protocols currently available for ChIP analysis. Unfortunately, many of these kits and protocols are specific to one lab and a confined number of cell types. The truChIP™ reagents and protocols developed by scientists at Covaris, in partnership with several prominent laboratories across the globe, have universal application across all mammalian cell types and tissues. Many ChIP protocols require time consuming and tedious optimization of multiple parameters, most notably chromatin shearing steps. Unfortunately, standard probe and bath sonicators demonstrate large variability from sample-to-sample, and day-to-day, even with the same researcher conducting the experiments, making it impossible to transfer protocols. With Covaris’ truChIP™ Chromatin Shearing kits minimal optimization is required by an end-user to outline the parameters best suited for specific antibodies (e.g. time of fixation). Combined with the high performance “clinical-grade” reproducibility of the Covaris focused-ultrasonicators, once optimal protocol parameters are determined, they can be reliably shared with coworkers and collaborators with consistent reproducible results.

FIGURE 7: Efficient shearing of chromatin from different tissues

FIGURE 7: Chromatin sheared from multiple tissue samples following the standard truChIP Tissue Chromatin Shearing Kit with SDS Shearing Buffer analyzed on Bioanalyzer™ yield identical shearing length profiles.

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The truChIP Chromatin Shearing Kits are available in multiple formats to provide the flexibility you need to get the best results possible in your ChIP experiments. Many standard “home-brew” ChIP protocols and commercially available ChIP kits require long formaldehyde fixation times and high SDS concentrations in order to overcome the limitations of probe and bath sonicators. Both over-fixation and high SDS concentrations can damage epitopes and adversely affect immunoprecipitation of protein:DNA complexes. Benefiting from the isothermal and controlled Covaris focused-ultrasonicators utilizing AFA, short fixation times (5 min or less) and shearing in non-ionic, or low SDS (0.1%) shearing buffer are now possible with the truChIP Chromatin Shearing Kits. The flexibility in fixation time allows the proper fixation for your epitopes to be determined empirically. Additionally, chromatin sheared in non-ionic or low SDS shearing is more compatible with downstream immunoprecipitation and analytical methods.

FIGURE 8: Flexible protocol can be optimized for ChIP with more than one antibody simultaneously

FIGURE 8: 2×106 of MS4221 cells were cross-linked with 1% fresh formaldehyde for 5, 10, and 30 minutes. The chromatin from the cross-linked cells was sheared by 10 minutes of AFA in a Covaris Focused-ultrasonicator. Aliquots representing sheared chromatin from ~5×105 cells were used for ChIP analysis with anti-ubiquityl H2B and Suz12 antibodies; 5ng of DNA from each IP was used for qPCR of the GAPDH and Hox1A promoters respectively. The fold enrichment was calculated by comparing the ubiquityl-Histone H2B and Suz12 qPCR results to input DNA. The 5 minute formaldehyde cross-linking provides robust enrichment of both the ubiquityl-Histone H2B at the GAPDH promoter and Suz12 transcription factor at the Hox1A promoter. This demonstrates the flexibility of the truChIP Chromatin Shearing protocol to easily accommodate ChIP analysis of both high and low abundance protein DNA interactions using one set of parameters.

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Chromatin Shearing Kits
truChIP™ High Cell Chromatin Shearing Kit with SDS Shearing Buffer
truChIP™ High Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer
truChIP™ Low Cell Chromatin Shearing Kit with SDS Shearing Buffer
truChIP™ Low Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer
truChIP™ Tissue Chromatin Shearing Kit with SDS Shearing Buffer

AFA™ Ultrasonicators:
M220 Focused-ultrasonicator
S220 Focused-ultrasonicator
E220 Focused-ultrasonicator
LE220 Focused-ultrasonicator

CryoPrep™ Advanced Tissue Processing System
CryoPrep System
tissueTUBE Devices

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Chromatin Shearing
truChIP™ High Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer
truChIP™ High Cell Chromatin Shearing Kit with SDS Shearing Buffer
truChIP™ Low Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer
truChIP™ Low Cell Chromatin Shearing Kit with SDS Shearing Buffer
truChIP™ Tissue Chromatin Shearing Kit with SDS Shearing Buffer

truChIP™ Chromatin Shearing Kit MSDS Information

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Selected References

Repression of Androgen Receptor Transcription through the E2F1/DNMT1 Axis. Conrad David Valdez, Joanne N. Davis, Hana M. Odeh, Tristan L. Layfield, Craig S. Cousineau, Thomas R. Berton, David G. Johnson, Kirk J. Wojno, Mark L. Day. PLoS One. 2011; 6(9): e25187. Published online 2011 September 26. doi: 10.1371/journal.pone.0025187. PMCID: PMC3180375
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180375/?tool=pmcentrez

Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice. Harri Lempiäinen, Arne üller, Sarah Brasa, Soon-Siong Teo, Tim-Christoph Roloff, Laurent Morawiec, Natasa Zamurovic, Axel Vicart, Enrico Funhoff, Philippe Couttet, Dirk Schübeler, Olivier Grenet, Jennifer Marlowe, Jonathan Moggs, Rémi Terranova. PLoS One. 2011; 6(3): e18216. Published online 2011 March 24. doi: 10.1371/journal.pone.0018216. PMCID: PMC3063791
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063791/?tool=pmcentrez

Efficient Double Fragmentation ChIP-seq Provides Nucleotide Resolution Protein-DNA Binding Profiles. Michal Mokry, Pantelis Hatzis, Ewart de Bruijn, Jan Koster, Rogier Versteeg, Jurian Schuijers, Marc van de Wetering, Victor Guryev, Hans Clevers, Edwin Cuppen. PLoS One. 2010; 5(11): e15092. Published online 2010 November 30. doi: 10.1371/journal.pone.0015092. PMCID: PMC2994895
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996544/?tool=pmcentrez

Stable S/MAR-based episomal vectors are regulated at the chromatin level. Federico Tessadori, Kang Zeng, Erik Manders, Martijn Riool, Dean Jackson, Roel van Driel. Chromosome Res. 2010 November; 18(7): 757-775. Published online 2010 November 16. doi: 10.1007/s10577-010-9165-4. PMCID: PMC2996544
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996544/?tool=pmcentrez

ZBED6, a Novel Transcription Factor Derived from a Domesticated DNA Transposon Regulates IGF2 Expression and Muscle Growth. Ellen Markljung, Lin Jiang, Jacob D. Jaffe, Tarjei S. Mikkelsen, Ola Wallerman, Martin Larhammar, Xiaolan Zhang, Li Wang, Veronica Saenz-Vash, Andreas Gnirke, Anders M. Lindroth, Romain Barrés, Jie Yan, Sara Strömberg, Sachinandan De, Fredrik Pontén, Eric S. Lander, Steven A. Carr, Juleen R. Zierath, Klas Kullander, Claes Wadelius, Kerstin Lindblad-Toh, Göran Andersson, Göran Hjälm, Leif Andersson. PLoS Biol. 2009 December; 7(12): e1000256. Published online 2009 December 15. doi: 10.1371/journal.pbio.1000256. PMCID: PMC2780926
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2780926/?tool=pmcentrez

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