Features & Benefits
|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|
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: Reproducible size specific chromatin shearing
FIGURE 5: 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 6: Efficient shearing of chromatin from different tissues
FIGURE 6: 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 7: Flexible protocol can be optimized for ChIP with more than one antibody simultaneously
FIGURE 7: 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.
|Description||Ultra-low Cell||Low Cell||High Cell||Batch Processing||Tissue ChIP||Native ChIP||FFPE ChIP|
|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||truChIP Native Chromatin Shearing Kit||truChIP FFPE Chromatin Shearing Kit|
|Input||<100,000 cells||1 to 3 M cells||5 to 30 M cells||50 to 200 M cells||20 to 120 mg||<1 M cells, up to 30 M cells||2×10 µm slides|
|Samples Processed/Kit||50||50||15||2||Dependent on tissue mass||25||25|
|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||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||0.13- 1 mL||130 µL|
|Link to Protocol||Protocol||Protocol||Protocol||Protocol||Protocol||Protocol||Protocol|
|Safety Data Sheet||Safety Data Sheets||Safety Data Sheets||Safety Data Sheets||Safety Data Sheets||Safety Data Sheets||NA||NA|
|Product PN without Formaldehyde*||520158*||520127*||520127*||520127*||520238*||NA||NA|
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
FFPE Chromatin Shearing Protocols
Cultured Cells ChIP Protocols
Covaris truChIP and AFA® with the Chromatrap®Bead-free Immunoprecipitation Technology
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.
- Kuznetsov VI, Haws SA, Fox CA, Denu JM. General method for rapid purification of native chromatin fragments. J Biol Chem. 2018. DOI: 10.1074/jbc.RA118.002984
- Tharp KM, Kang MS, Timblin GA, et al. Actomyosin-Mediated Tension Orchestrates Uncoupled Respiration in Adipose Tissues. Cell Metab. 2018;27(3):602-615.e4. DOI: 10.1016/j.cmet.2018.02.005
- Manni M, Gupta S, Ricker E, et al. Regulation of age-associated B cells by IRF5 in systemic autoimmunity. Nat Immunol. 2018;19(4):407-419. DOI:10.1038/s41590-018-0056-8
- Yao H, Hill SF, Skidmore JM, et al. CHD7 represses the retinoic acid synthesis enzyme ALDH1A3 during inner ear development. JCI Insight. 2018;3(4). DOI: 10.1172/jci.insight.97440
- Donnard E, Vangala P, Afik S, et al. Comparative Analysis of Immune Cells Reveals a Conserved Regulatory Lexicon. Cell Syst. 2018;6(3):381-394.e7. DOI: 10.1016/j.cels.2018.01.002
- Liang C, Wang S, Qin C, et al. TRIM36, a novel androgen-responsive gene, enhances anti-androgen efficacy against prostate cancer by inhibiting MAPK/ERK signaling pathways. Cell Death Dis. 2018;9(2):155. DOI: 10.1038/s41419-017-0197-y
- Park SM, Choi EY, Bae DH, Sohn HA, Kim SY, Kim YJ. The LncRNA EPEL Promotes Lung Cancer Cell Proliferation Through E2F Target Activation. Cell Physiol Biochem. 2018;45(3):1270-1283. DOI: 10.1159/000487460
- Josipovic I, Pflüger B, Fork C, et al. Long noncoding RNA LISPR1 is required for S1P signaling and endothelial cell function. J Mol Cell Cardiol. 2018;116:57-68. DOI: 10.1016/j.yjmcc.2018.01.015
- Kim M, Astapova II, Flier SN, et al. Intestinal, but not hepatic, ChREBP is required for fructose tolerance. JCI Insight. 2017;2(24). DOI: 10.1172/jci.insight.96703
- Ramaswamy K, Forbes L, Minuesa G, et al. Peptidomimetic blockade of MYB in acute myeloid leukemia. Nat Commun. 2018;9(1):110. DOI: 10.1038/s41467-017-02618-6
- Humblin E, Thibaudin M, Chalmin F, et al. IRF8-dependent molecular complexes control the Th9 transcriptional program. Nat Commun. 2017;8(1):2085. DOI: 10.1038/s41467-017-01070-w
- Bu Y, Yoshida A, Chitnis N, et al. A PERK-miR-211 axis suppresses circadian regulators and protein synthesis to promote cancer cell survival. Nat Cell Biol. 2018;20(1):104-115. DOI: 10.1038/s41556-017-0006-y
- Li L, Fan CM. A CREB-MPP7-AMOT Regulatory Axis Controls Muscle Stem Cell Expansion and Self-Renewal Competence. Cell Rep. 2017;21(5):1253-1266. DOI: 10.1016/j.celrep.2017.10.031
- Forsyth CB, Shaikh M, Bishehsari F, et al. Alcohol Feeding in Mice Promotes Colonic Hyperpermeability and Changes in Colonic Organoid Stem Cell Fate. Alcohol Clin Exp Res. 2017;41(12):2100-2113. DOI: 10.1111/acer.13519
- Isobe K, Jung HJ, Yang CR, et al. Systems-level identification of PKA-dependent signaling in epithelial cells. Proc Natl Acad Sci USA. 2017;114(42):E8875-E8884. DOI: 10.1073/pnas.1709123114
- Zheng X, Han H, Liu GP, et al. LncRNA wires up Hippo and Hedgehog signaling to reprogramme glucose metabolism. EMBO J. 2017;36(22):3325-3335. DOI: 10.15252/embj.201797609
- Osabe M, Tajika T, Tohkin M. Allopurinol suppresses expression of the regulatory T-cell migration factors TARC/CCL17 and MDC/CCL22 in HaCaT keratinocytes via restriction of nuclear factor-κB activation. J Appl Toxicol. 2018;38(2):274-283. DOI: 10.1002/jat.3522
- Hatanaka Y, Tsusaka T, Shimizu N, et al. Histone H3 Methylated at Arginine 17 Is Essential for Reprogramming the Paternal Genome in Zygotes. Cell Rep. 2017;20(12):2756-2765. DOI: 10.1016/j.celrep.2017.08.088
- Nathan S, Ma Y, Tomita YA, De oliveira E, Brown ML, Rosen EM. BRCA1-mimetic compound NSC35446.HCl inhibits IKKB expression by reducing estrogen receptor-α occupancy in the IKKB promoter and inhibits NF-κB activity in antiestrogen-resistant human breast cancer cells. Breast Cancer Res Treat. 2017;166(3):681-693. DOI: 10.1007/s10549-017-4442-y
- Mehta S, Cronkite DA, Basavappa M, et al. Maintenance of macrophage transcriptional programs and intestinal homeostasis by epigenetic reader SP140. Sci Immunol. 2017;2(9). DOI: 10.1126/sciimmunol.aag3160
- Bandyopadhaya A, Tsurumi A, Rahme LG. NF-κBp50 and HDAC1 Interaction Is Implicated in the Host Tolerance to Infection Mediated by the Bacterial Quorum Sensing Signal 2-Aminoacetophenone. Front Microbiol. 2017;8:1211. DOI: 10.3389/fmicb.2017.01211
- Gullicksrud JA, Li F, Xing S, et al. Differential Requirements for Tcf1 Long Isoforms in CD8 and CD4 T Cell Responses to Acute Viral Infection. J Immunol. 2017;199(3):911-919. DOI: 10.4049/jimmunol.1700595
- Gulchina Y, Xu SJ, Snyder MA, Elefant F, Gao WJ. Epigenetic mechanisms underlying NMDA receptor hypofunction in the prefrontal cortex of juvenile animals in the MAM model for schizophrenia. J Neurochem. 2017;143(3):320-333. DOI: 10.1111/jnc.14101
- Shan Q, Zeng Z, Xing S, et al. The transcription factor Runx3 guards cytotoxic CD8 effector T cells against deviation towards follicular helper T cell lineage. Nat Immunol. 2017;18(8):931-939. DOI: 10.1038/ni.3773
- Michaelson JJ, Shin MK, Koh JY, et al. Neuronal PAS Domain Proteins 1 and 3 Are Master Regulators of Neuropsychiatric Risk Genes. Biol Psychiatry. 2017;82(3):213-223. DOI: 10.1016/j.biopsych.2017.03.021
- Aldiri I, Xu B, Wang L, et al. The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis. Neuron. 2017;94(3):550-568.e10. DOI: 10.1016/j.neuron.2017.04.022
- Hwang JR, Chou CL, Medvar B, Knepper MA, Jung HJ. Identification of β-catenin-interacting proteins in nuclear fractions of native rat collecting duct cells. Am J Physiol Renal Physiol. 2017;313(1):F30-F46. DOI: 10.1152/ajprenal.00054.2017
- Hsieh LT, Nastase MV, Roedig H, et al. Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants. Int J Mol Sci. 2017;18(3). DOI: 10.3390/ijms18030595
- Hirose M, Hasegawa A, Mochida K, et al. CRISPR/Cas9-mediated genome editing in wild-derived mice: generation of tamed wild-derived strains by mutation of the a (nonagouti) gene. Sci Rep. 2017;7:42476. DOI: 10.1038/srep42476
- Li C, Wang S, Xing Z, et al. A ROR1-HER3-lncRNA signalling axis modulates the Hippo-YAP pathway to regulate bone metastasis. Nat Cell Biol. 2017;19(2):106-119. DOI: 10.1038/ncb3464
- Zhang J, Vlasevska S, Wells VA, et al. The CREBBP Acetyltransferase Is a Haploinsufficient Tumor Suppressor in B-cell Lymphoma. Cancer Discov. 2017;7(3):322-337. DOI: 10.1158/2159-8290.CD-16-1417
- Grajkowska LT, Ceribelli M, Lau CM, et al. Isoform-Specific Expression and Feedback Regulation of E Protein TCF4 Control Dendritic Cell Lineage Specification. Immunity. 2017;46(1):65-77. DOI: 10.1016/j.immuni.2016.11.006
- Fork C, Vasconez AE, Janetzko P, et al. Epigenetic control of microsomal prostaglandin E synthase-1 by HDAC-mediated recruitment of p300. J Lipid Res. 2017;58(2):386-392. DOI: 10.1194/jlr.M072280
- Bersaas A, Arnoldussen YJ, Sjøberg M, Haugen A, Mollerup S. Epithelial-mesenchymal transition and FOXA genes during tobacco smoke carcinogen induced transformation of human bronchial epithelial cells. Toxicol In Vitro. 2016;35:55-65. DOI: 10.1016/j.tiv.2016.04.012
- Song I, Kim K, Kim JH, et al. GATA4 negatively regulates osteoblast differentiation by downregulation of Runx2. BMB Rep. 2014;47(8):463-8. DOI: 10.1002/jbm4.10027
- Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells. Zuklys S, et al.Nature Immunology, 2016. DOI: 10.1038/ni.3537
- Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity. Xing S, et al. Nature Immunology, 2016. DOI: 10.1038/ni.3456
- miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis. Xu Z, et al. Nature Communications, 2016. DOI: 10.1038/ncomms11422
- cChIP-seq: a robust small-scale method for investigation of histone modifications. Valensisi C, et al. BMC Genomics, 2015. DOI: 10.1186/s12864-015-2285-7
- Nrf1 and Nrf2 Transcription Factors Regulate Androgen Receptor Transactivation in Prostate Cancer Cells. Schultz MA, et al. PLoS ONE, 2014. DOI: 10.1371/journal.pone.0087204
- Bivalent chromatin marks developmental regulatory genes in the mouse embryonic germline in vivo. Sachs et al. Cell Reports, 2013. DOI: 10.1016/j.celrep.2013.04.032
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.
Covaris provides tools and technologies to improve pre-analytical sample preparation, enable novel drug formulations, and manage compounds in the drug discovery process.