|Developed for low cell inputs and tissue samples|
|Easy optimization for new cell lines or epitopes of interest|
|Standardized workflow reduces lab to lab variability and delivers the highest reproducibility|
|Increased sensitivity, discover epitopes that were previously not observed|
|The solution for high-throughput automated epigenomics|
|Validated solution for all mammalian cells with truChIP Kits|
The use of AFA is pervasive in the chromatin sample preparation space. Below, a brief description of each application is provided with reference to key publications that can be viewed for additional information.
|ChIP-Seq||ChIP-Seq is a powerful sequencing technique used to profile DNA and protein interactions. ChIP-Seq enables scientists to understand key mechanisms of gene regulation, replication, and DNA repair 1, 2, 3, 4, 5.|
|ChIP-ChIP||Chromatin IP is done using an antibody against a protein of interest. The DNA and input samples are purified, fluorescently tagged, and subsequently hybridized to a microarray for analysis 6.|
|ChIRP-Seq||ChIRP (chromatin isolation by RNA purification) is a technique used for studying the genomic binding sites of chromatin-associated with lncRNAs 7.|
|Hi-C||Hi-C is used to study the overall genome structure and biophysical properties of chromatin and the long-range contacts between distant genomic elements such as genes and regulatory elements. Hi-C provides a unique and powerful tool to study nuclear organization and chromosome architecture 8.|
|ChIP-qPCR||ChIP-qPCR can be used to study regulatory regions of interest if the genomic binding sites are known. Using ChIP-qPCR, the level of enrichment can be calculated at a specific loci 9.|
|Methylation Sequencing||DNA methylation is an epigenetic modification made directly to DNA that can alter the state of genes and change expression levels. DNA methylation analysis is accomplished by treating DNA with bisulfite to convert unmethylated cytosine residues to uracil for both capture-based Methyl-Seq (e.g., amplicon-based and target enrichment) and whole genome bisulfite sequencing (WGBS). DNA can be extracted from a variety of sources such as cultured cell lines 10, 11. Focused acoustic shearing by Covaris is part of the most widely used DNA methylation analysis protocols including: Illumina whole-genome bisulfite sequencing for Methylation Analysis, NuGEN Methyl-Seq Library Systems, Agilent SureSelect Methyl-Seq Target Enrichment for Illumina.|
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.
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.
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.
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.
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.
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.
truChIP – Mammalian Cell ChIP
The Covaris truChIP Chromatin Shearing Kit was developed and optimized using mammalian cells. truChIP is guaranteed to work with all mammalian cells and the conditions can be optimized in one week or less. AFA has demonstrated to provide highly reproducible results without damaging protein epitopes. Moreover, because AFA is a highly controlled mechanical shearing technology, more than ~70% of fragments are sheared to the correct size range for sequencing. Pairing AFA with truChIP reagent kits provides investigators the flexibility to process both low (as low as ~10K cells) and high cell (up to 200M cells) inputs. Chromatin samples prepared using truChIP are compatible with commercially available antibodies for immunoprecipitation (IP) and all subsequent steps of the sample preparation workflow.
truChIP – Tissue ChIP
The Covaris truChIP Chromatin Shearing Tissue Kit can be used in combination with the Covaris CP02 cryoPREP Pulverizer to process tissue samples for ChIP-Seq. Covaris developed the protocol using liver, brain, and muscle tissue from mice. The truChIP Chromatin Shearing Tissue Kit is optimized for efficient and reproducible chromatin shearing when working with a variety of hard and soft tissue types.
|Description||Ultra-low Cell Protocol||Low Cell Protocol||High Cell Protocol||Batch Processing Protocol||Tissue ChIP Protocol|
|Product Name||truChIP Ultra-Low Chromatin Shearing Kit with Formaldehyde||truChIP Chromatin Shearing Kit with Formaldehyde||truChIP Chromatin Shearing Kit with Formaldehyde||truChIP Chromatin Shearing Kit with Formaldehyde||truChIP Chromatin Shearing Tissue Kit|
|Input||<100,000 cells||1 to 3 Million cells||5 to 30 Million cells||50 to 200 Million cells||20 to 120 mg|
|Samples Processed/Kit||50||50||15||2||Dependent on tissue mass|
|AFA Consumable||microTUBE – 130||microTUBE–130||milliTUBE–1 mL with AFA fiber||6 × milliTUBE–1 mL with AFA fiber||microTUBE-130 or milliTUBE–1 mL with AFA fiber|
|Processing Volume||130 µL||130 µL||1 mL||6 x 1 mL||0.13-1 mL|
|Link to Protocol||Protocol||Protocol||Protocol||Protocol||Protocol|
|Safety Data Sheet||Safety Data Sheets||Safety Data Sheets||Safety Data Sheets||Safety Data Sheets||Safety Data Sheets|
|Product PN without Formaldehyde*||520158*||520127*||520127*||520127*||520238*|
|Part Number||Item Name||Description|
|520156||truChIP Ultra-Low Chromatin Shearing Kit||Includes reagents required to run ultra-low cell (<100k) protocol. AFA processing tubes are sold separately (see required parts & accessories table).|
|520154||truChIP Chromatin Shearing Kit||Includes reagents required to run the chromatin shearing protocol to process up to 200M cells. AFA processing tubes are sold separately (see required parts & accessories table).|
|520237||truChIP Chromatin Shearing Tissue Kit||Includes reagents required to process both low mass inputs (20 to 50 mg in the microTUBE–130 µL) and high mass inputs (up to 120 mg in milliTUBE–1 mL with AFA fiber). AFA processing tubes are sold separately (see required parts & accessories table).|
|500001||CP02 Cryoprep Pulverizer||Tissue pulverizer to cryofracture tissue and disrupt matrix for downstream applications including tissueChIP.|
|500497||CP02 Low and High Tissue Mass Starter Kit||Includes parts and accessories to process low (<50 mg to 1 g) and high (1 to 2 g) tissue samples for downstream applications including tissue ChIP.|
Required Parts & Accessories
Holder & Insert
|ME220 Holder & Insert||S-Series Holder||E220 Evolution Rack||E220 Rack||LE220 Rack|
|520045||microTUBE AFA Fiber Pre-Slit|
|520052||microTUBE AFA Fiber Crimp-Cap||NA||500514|
|520216||microTUBE-130 AFA Fiber Screw-Cap||500414|
|520053||8 microTUBE Strip V1||NA||500514|
|520217||8 microTUBE-130 AFA Fiber Strip V2||NA||500518|
|520078||96 microTUBE Plate||NA||NA||NA||NA||Not Required||500329|
|520130||milliTUBE–1 mL with AFA fiber||500414|
Tissue ChIP Protocols
*PN 520083 has been replaced with PN 520237
Cultured Cells ChIP Protocols
Peer-reviewed publications citing Covaris has grown considerably over the past five years. In 2016, there were over 500 papers that cited using truChIP, Covaris ultrasonicators, or a combination of both for chromatin sample prep. Below, a handful of high impact papers were selected to be included here. Importantly, the papers referenced below highlight some of the many key advantages provided by AFA, such as low cell input methods.
- Bivalent chromatin marks developmental regulatory genes in the mouse embryonic germline in vivo. Sachs et al. Cell Reports, 2013.
Focus: In this paper, the authors developed a low cell (<~10K) protocol for studying the epigenetic landscape in mouse primordial germ cells (PGC) with a specific focus on how genes marked as bivalent affect development. Importantly, the protocol developed here did not require a carrier or preamplification step to analyze histone marks in PGCs.
- Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells. Zuklys S, et al. Nature Immunology, 2016.
Focus: Using ChIP-Seq, the authors studied the regulatory action and identified target genes of Foxn1 in thymic epithelial cell differentiation.
- Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity. Xing S, et al. Nature immunology, 2016.
Focus: This paper focused on the functional role of transcription factors (Tcf1 and Lef1) that play an instructive role in maintaining immune appropriate cell identity.
- Nrf1 and Nrf2 Transcription Factors Regulate Androgen Receptor Transactivation in Prostate Cancer Cells. Schultz MA, et al. PLoS ONE, 2014.
Focus: Studied key transcription factors (Nrf1 and Nrf2) that regulate androgen receptor (AR) transactivation in castration resistant prostate cancer (CRPC) pathways that may be involved in conferring resistance to treatment.
- cChIP-seq: a robust small-scale method for investigation of histone modifications. Valensisi C, et al. BMC Genomics, 2015.
Focus: Studied key histone modifications (H3K4me3, H3Kme1, H3K27me3 and H3K4me1) using a low cell input (~10K cells) method.
- Chromatin immunoprecipitation and microarray-based analysis of protein location. Lee TI, et al. Nature protocols, 2006.
Focus: Provides a protocol for genome-wide location analysis using ChIP-ChiP. This technique combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions. This paper provides a high-level overview of this application and technical challenges associated with the workflow.
- Rapid evolutionary turnover underlies conserved lncRNA–genome interactions. Quinn JJ, et al. Genes & Development, 2016.
Focus: Using Drosophila, genomic binding sites of chromatin associated lncRNAs (roX1 and rox1) were studied to further understand the regulatory influences the non-coding genome can have on the chromatin state.
Focus: This paper provides an overview of the Hi-C technique. Hi-C is used to study the overall genome structure and biophysical properties of chromatin and the long-range contacts between distant genomic elements such as genes and regulatory elements.
- miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis. Xu Z, et al. Nature Communications, 2016.
Focus: Evaluated the molecular mechanisms of apoptotic pathways by focusing on a key transcription factor -CHOP/GADD153 and actions of the microRNA, miR-216b.
Focus: Kulis, et al. used whole genome bisulfite sequencing (WGBS) to profile the methylation status in B-cells.
11. Rackham OJL, Langley SR, Oates T, et al. A Bayesian Approach for Analysis of Whole-Genome Bisulfite Sequencing Data Identifies Disease-Associated Changes in DNA Methylation. Genetics. 2017;205(4):1443-1458. doi:10.1534/genetics.116.195008.
Focus: In this paper, the authors studied how DNA methylation patterns at specific genomic loci explain the pathogenesis glomerulonephritis. The author also provide details on the Bayesian analysis used for analyzing the WGBS data.
Selected Covaris Application Notes
- Optimized protocol for robust chromatin shearing and immunoprecipitation of human pancreatic islets using the Covaris Focused-ultrasonicator Escalada et al.
Focus: In this application note, Escalada et al. processed human islet cells for ChIP-Seq and ChIP-qPCR using the S220 Focused-ultrasonicator. The authors developed a protocol using the focused acoustics shearing platform to compare its performance to other traditional methods including: probe and water bath sonication. Pre-sequencing fragment size analysis data is presented as well as ChIP-qPCR and ChIP-Seq results.
- Preparation of whole body Caenorhabditis elegans extracts for chromatin immunoprecipitation using the Covaris® S220 Focused-ultrasonicator Esse et al.
Focus: Ruben et. al. from Boston University School of Medicine developed a ChIP-Seq sample preparation protocol for whole body C. elegans extracts. The authors of this application note provide a step-by-step sample preparation workflow and highlight the key advantages of using the Covaris S220 Focused-ultrasonicator.
- Streamlined Ultra Low Sample Input and Processing Volume Chromatin Shearing Protocols for Fly Embryos and Mammalian Cell Lines Baghdadi et al.
Focus: Here, the authors developed low volume (from 20 to 50 μl) protocols using truChIP for both mammalian cell lines and fly embryos. In this application note, the working conditions for processing down to ~10,000 mammalian cells and 5 stage-17 Drosophila embryos are outlined. As a result, these working conditions can be adopted by investigators seeking to perform both low cell counts and volume ChIP experiments.
Focus: In this application note using yeast cells from S. cerevisiae, the research team at the MIT Broad Institute developed a highly reproducible sample preparation protocol for ChIP-Seq. To compare the results, this group processed samples in parallel using a Branson Sonifier for a true side-by-side comparison. At the conclusion of the study, it was noted that AFA provided the consistency and reproducibility needed for NGS library preparation and scalability with less hands on time.
- Optimizing sample fixation and chromatin shearing for improved sensitivity and reproducibility of chromatin immunoprecipitation Khoja et al.
Focus: This application note provides an overview of Covaris the truChIP reagent kit paired with AFA. Here, using MS4221 lymphoblast cells, the authors demonstrate the importance of good sample preparation practices for successful ChIP analysis. In this note, the authors demonstrate how AFA delivers precise control during processing to provide more chromatin available for the immunoprecipitation, preserve protein epitopes, and recover high-quality DNA for sequencing.
From the 2012 AGBT conference, this poster provides users an overview of AFA and how it can be integrated into the chromatin sample preparation workflow for NGS.
Optimizing ChIP Sample Preparation for Reproducibility
Presenter: Hamid Khoja, PhD, Covaris Principal Scientist
Focus: In this Covaris sponsored Bitesize Bio webinar, a focus on how to optimize sample preparation for mammalian cells ChIP is explained. A high-level overview of AFA and key advantages provided over other mechanical and enzymatic-based shearing methods is discussed.
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 20-120 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 as 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 to process mammalian cells.
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.
Covaris provides tools and technologies to improve pre-analytical sample preparation, enable novel drug formulations, and manage compounds in the drug discovery process.