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Abstract

Protocol

Representative Results

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Materials

References

Biology

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Published: May 1st, 2013

DOI:

10.3791/50064

1Department of Pathology, University of Utah School of Medicine

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.

Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.

This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

1. Isolation of Mouse Splenic Naïve CD4 T Cells

  1. Sacrifice the mouse in a humane manner consistent with Institutional Animal Care and Use Committee (IACUC) protocols. Dissect the spleen and place it in Petri dish containing 10 ml of DMEM with 10% FBS.
  2. Crush the spleen using frosted ends of two glass slides to release the splenocytes. Transfer the cell suspension in a 15 ml conical tube.
  3. Collect the cells by centrifugation at 200 x g (~1,200 rpm for a clinical centrifuge wit.......

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The Chromatin Immunoprecipitation (ChIP) protocol presented here controls for differences, if any, in the amount of DNA used in PCR through use of a primer pair that amplifies an unbound region of genome, thus serving as a "loading control". In the example shown in Figure 3, we have used the coding region of the mouse Actb gene as a region unbound to our protein of interest and the transcription factor NFAT binding site on mouse Il2 promoter as target region. Alternatively, a regio.......

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The protocol above provides a robust method of accurately quantifying DNA enrichment from primary lymphocytes using ChIP. One major reason for robustness in this protocol is the inclusion of biological replicates. The above protocol uses three replicates, the enrichment for which is calculated independently. The outputs are then averaged to provide a degree of enrichment and standard deviations calculated to provide a measure of variability. For each sample, three technical replicates are also performed to elimina.......

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This work was supported by NIH grants CA141009 and GM39067. We thank E. Parnell and R. Yarrington for comments on the written portion.

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Name Company Catalog Number Comments
Name of the reagent Company Catalog number Comments
Formaldehyde Sigma F-8775 Store at RT
Phosphate Buffered Saline Hyclone SH30256.01 Store at 4 °C
Protease Inhibitor tablets Roche 04693116001 Store at 4 °C
Protein G magnetic beads Active Motif 101945 Store at 4 °C
RNase A (20 mg/ml) EMD Millipore 556746 Store at -20 °C
Proteinase K (20 mg/ml) Roche 03115879001 Dissolve in 50 mM Tris-HCl, 10 mM CaCl2, pH 8.0
Platinum Taq DNA Polymerase Invitrogen 10966-034 Store at -20 °C
SYBR Green I Invitrogen S7567 Store at -20 °C
1 M Glycine Store at RT
Cell lysis buffer (5 mM Pipes, pH 8.0; 85 mM KCl; 0.5% NP-40) Store at 4 °C
Nuclear lysis buffer (50 mM Tris, pH 8.1; 10 mM EDTA; 1% SDS) Store at RT
ChIP dilution buffer (0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris pH 8.1; 190 mM NaCl) Store at 4 °C
Low salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris pH 8.1; 150 mM NaCl) Store at 4 °C
High salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris pH 8.1; 600 mM NaCl) Store at 4 °C
LiCl wash buffer (0.25 M LiCl; 1% NP-40; 1% Sodium Deoxycholate, 1 mM EDTA; 10 mM Tris pH 8.0) Store at 4 °C
TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA) Store at 4 °C
Elution buffer (1% SDS; 0.1 M NaHCO3) Prepare fresh
5 M NaCl Store at RT
0.5 M EDTA Store at RT
1 M Tris-HCl, pH 6.5 Store at RT
Table of Specific Reagents
Clay Adams Brand Nutator Becton Dickinson Model: 421105
Magnetic Stand Promega Z5342
Qiaquick PCR Purification Kit Qiagen 28106
Masonix Sonicator 3000 QSonica Model: S3000
UV Spectrophotometer NanoDrop Technologies ND-1000
Heating Block VWR 13259-030
Rotator VWR 80085-692
Refrigerated bench top centrifuge Beckman Coulter Model: Allegra X-12R
Microcentrifuge Eppendorf 5415 D
Table of Equipment

  1. Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q., Farnham, P. J. Use of chromatin immunoprecipitation to clone novel E2F target promoters. Molecular and Cellular Biology. 21, 6820-6832 (2001).
  2. Weinmann, A. S., Farnham, P. J. Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods. 26, 37-47 (2002).
  3. Li, Q., et al. Constitutive nuclear localization of NFAT in Foxp3+ regulatory T cells independent of calcineurin activity. J. Immunol. 188, 4268-4277 (2012).
  4. Shakya, A., Kang, J., Chumley, J., Williams, M. A., Tantin, D. Oct1 is a switchable, bipotential stabilizer of repressed and inducible transcriptional states. The Journal of Biological Chemistry. 286, 450-459 (2011).
  5. Dahl, J. A., Collas, P. A rapid micro chromatin immunoprecipitation assay (microChIP). Nature Protocols. 3, 1032-1045 (2008).
  6. Lubelsky, Y., Macalpine, H. K., Macalpine, D. M. Genome-wide localization of replication factors. Methods. 57, 187-195 (2012).
  7. O'Neill, L. P., VerMilyea, M. D., Turner, B. M. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nature Genetics. 38, 835-841 (2006).
  8. Sikes, M. L., et al. A streamlined method for rapid and sensitive chromatin immunoprecipitation. Journal of Immunological Methods. 344, 58-63 (2009).
  9. Rhee, H. S., Pugh, B. F. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell. 147, 1408-1419 (2011).
  10. Rhee, H. S., Pugh, B. F. Genome-wide structure and organization of eukaryotic pre-initiation complexes. Nature. 483, 295-301 (2012).
  11. Kharchenko, P. V., Tolstorukov, M. Y., Park, P. J. Design and analysis of ChIP-seq experiments for DNA-binding proteins. Nature Biotechnology. 26, 1351-1359 (2008).
  12. Robertson, G., et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nature Methods. 4, 651-657 (2007).

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