These protocols have been optimized for both mammalian cell lines and fly embryos and reliably fragment chromatin in low volumes (from 20 to 50 μl) from down to 10,000 mammalian cells and 5 stage-17 Drosophila embryos. They provide a good starting point when aiming to develop and optimize reduced cell and volume chromatin shearing protocols for ChIP experiments.
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
How should I collect adherent cells and perform sample quantification?
There are a number of different methods that can be employed to quantify the number of adherent cells. Typically, one can culture an additional plate, trypsinize the cells, followed by counting. The counts should be consistent with the other culture plates started at the same time.
Why do I need to run a fixation time course? What cell types is it most critical for and why?
Fixation is not only cell line dependent, but it is also epitope dependent. Not all cells fix at the same rate, therefore, it is imperative to carry out both a one-time fixation and shearing time course to establish a protocol for cell lines and tissue types. Importantly, certain proteins can be quite sensitive to formaldehyde fixation resulting in conformational changes from over-fixation that render them unrecognizable by an antibody.
Why is the methanol-free formaldehyde provided with truChIP kits recommended over other fixing solutions?
Many formaldehyde reagents contain methanol as a stabilizing agent to prevent oxidation and polymerization. Based on our evaluations, we have shown that the presence of methanol leads to the generation of large molecular weight chromatin complexes resistant to shearing.
Why is the fixation time in the Covaris truChIP protocol so short?
In stark contrast to Covaris AFA, bath and probe sonicators utilize up to 150× more energy. As a result, these other methods demand longer fixation times in order to minimize damage to the chromatin. Covaris AFA is a more efficient and gentle mechanical shearing technology that does not require long fixation times.
Why do we need to run a shearing time course if we have already done ChlP before?
Unfortunately, not all cells shear to the same size range at the same rate. As a result, Covaris recommends users to carry out a shearing time course to empirically determine the optimal treatment conditions. For ChIP-seq, it is important to optimize the chromatin shearing size range to be compatible with the selected library preparation kit and sequencing platform. For Illumina platforms, it is recommended to shear chromatin to an average fragment size of 200-250 bp with a distribution up to ~700 bp.
What type of cells can truChIP kit be used for?
truChIP has been successfully tested with hundreds of mammalian cells lines by laboratories across the world. The truChIP kits are considered universal sample preparation kits guaranteed to work with all mammalian cells.
What is the lowest cell inputs that have been tested?
The truChIP ultra-low cell (Covaris PN: 520156) chromatin shearing kit has been used with as little as 10,000 cells.
I want to process 50M cells/sample–is it possible?
Yes, our milliTUBE–1 mL with AFA fiber (Covaris PN: 520135) can accommodate up to 30 million cells. Additionally, one can use the milliTUBE–2 mL (Covaris PN: 520132) for samples with higher inputs up to 60 million cells. Please note: the milliTUBE–2 mL is only compatible with the S220 and E220 platforms.
Does Covaris provide a protocol for tissue ChIP?
Yes, Covaris provides a truChIP kit (Covaris PN: 520083) for processing tissue masses within the range of 18-150 mg. To review this sample prep workflow, please click here.
Is it possible to fragment native ChIP using a Covaris?
Because the energy can be tightly controlled, native ChIP sample prep can be performed using AFA. Please contact us for further guidance on this particular application.
Can you simultaneously lyse and fragment in one tube?
Generally, it is not possible to lyse and shear chromatin at the same time in the same tube. One step methods require high energy processing with high detergent concentrations. The yield of those methods tends to be significantly lower than two step methods. For very low cell numbers, it is possible to do a one-step lysis and shearing as in our truChIP ultra low cell chromatin shearing kit.
May I use my own buffers and reagents?
Most chromatin sample preparation and shearing buffers were developed and optimized for use with bath or probe sonicators. Since Covaris AFA is a more advanced technology, off-label buffers are not compatible with Covaris AFA. Therefore, we strongly recommend customers to use the buffers provided with the truChIP kit since they have been validated with Covaris platforms.
May I combine your protocol with my other commercial protocols?
The truChIP kits are compatible with downstream IP protocols. Covaris provides the formulation of the buffers to ensure the IP step can be performed with any homebrew or commercial IP kit. For example, the truChIP chromatin shearing reagent kit shearing buffer for use with cells contains 1 mM EDTA, 10 mM Tris-HCl pH 7.6, 0.1% SDS.
How does having reduced SDS concentrations help with the Covaris kit?
Reduced SDS concentrations enables the utilization of more sheared chromatin in each IP. Additionally, SDS is a potent solubilizer. Accordingly, shearing with high concentrations of SDS can solubilize a significant amount of the desired epitope.
Is there a required RNase treatment?
Covaris recommends RNase treatment because the presence of RNA can potentially skew the smear analysis performed on the Bioanalyzer and agarose gels.
Why am I not seeing a gradual reduction in fragment size distribution as I shear my samples?
There are a few possibilities why no shearing is observed:
The cells were processed using reagents and protocols not optimized for use with AFA
The cells are being severely over-fixed
The crosslinked sheared chromatin has not been reversed or purified properly for DNA size range analysis
I optimized protocols for a cell line and now observing inconsistent shearing results. Why?
To ensure consistent results are achieved, Covaris recommends using the truChIP kit and to keep the cell numbers within the range outlined in the protocol. Any modifications made to the buffers and/or reagents provided, or deviations in the protocol, may lead to inconsistent shearing profiles.
Why did my IP fail?
IP can fail for a myriad of reasons. Some of the most common ones are:
Over shearing resulting in epitope damage
Fixation was not optimized for the protein of interest–the crosslinking was not sufficient
Incorrect IP buffer that has not been optimized for use with the selected antibody
Is it true that the fixation time cannot be reduced if a target of interest is not abundant?
No, the target abundance is independent of formaldehyde fixation–there is no direct correlation between fixation time and epitope abundance.
I have a large protein complex. What methods are recommended for these sample types?
For protein complexes, depending on the proximity of the protein of interest to the DNA, Covaris recommends the use of a dual fixation strategy. Specifically, this involves fixing proteins to proteins first with DSS, DGS, and DMA followed by fixing the proteins to DNA with formaldehyde.
If I fix shorter less/time, will I lose some downstream sensitivity?
Since fixation is cell line dependent and epitope dependent, it is certainly possible that the fixation might be too little or too much which can affect IP efficiency. Thus, this is why it is essential to carry out dual time course studies. Additionally, we recommend running a Western blot to check the epitope integrity during the shearing time course.
What is the average processing time per sample using truChIP?
Unfortunately, not all cells shear to the desired size with the same treatment time. Therefore, Covaris recommend that you carry out a fixation and a shearing time course to determine the optimal conditions.
Why do I need to control the temperature?
Formaldehyde fixation is reversible with temperature. If temperature is not controlled during chromatin shearing, undesired reverse crosslinking may occur leading to epitope loss.
Specifically, how do the duty factor (DF), power, and duration impact the fragment length?
For chromatin shearing using truChIP, Covaris has already optimized the duty factor (DF), peak incidence power (PIP), and cycles per burst (CPB). Therefore, the user is only responsible for optimizing the shearing time for the cell line or tissue sample.
May I use the truChIP kit with the M220 focused ultrasonicator?
Yes, all Covaris platforms are compatible for use with truChIP kits.
Yes, Covaris provides the milliTUBE–2 mL which can be used to shear chromatin in 2 mL volumes on the S and E-series instruments only. By using these tubes, one can double the number of cells and volume. For sample treatment settings, the duty factor (DF) will be doubled (compared to 1 mL protocol) and the processing times will increase.
What settings should I use on the Covaris for ChIRP/Hi-C, etc.?
ChIRP and Hi-C protocols have not been optimized in-house, but we do have customers who are using it. Please contact us for further guidance on this particular application.