An H3K27me3 Antibody with Reliable Results in ChIP-seq and CUT&RUN
Trimethylation on histone 3 lysine 27 (H3K27me3) is one of the most widely studied histone post-translational modifications (PTMs) in epigenetics research. Associated with gene inactivation, this modification is connected with diverse human diseases, including cancer1 and neurodegeneration2. The ability to precisely map the localization of H3K27me3 via ChIP-seq (Chromatin Immunoprecipitation Sequencing) or CUT&RUN (Cleavage Under Targets and Release Using Nuclease)3-5 is essential to understand how this PTM regulates chromatin structure and gene expression in disease. Although these assays differ in their resolution, cost, and cell input requirements, there is one reagent that is crucial to both assays: a highly specific H3K27me3 antibody.
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SNAP-ChIP Spike-Ins and Antibodies
EpiCypher is a leader in providing best-in-class antibodies for chromatin profiling experiments. Our SNAP-ChIP® technology uses panels of DNA-barcoded modified recombinant nucleosomes as spike-in controls to determine antibody specificity and enrichment directly in the context of a ChIP-seq experiment6, 7. These experiments6 have revealed two major truths in the field of chromatin antibodies:
1. Many antibodies display off-target binding
2. A nucleosome substrate is completely required for identifying highly specific antibodies for downstream chromatin profiling experiments
To address these concerns and enable high-quality ChIP studies, EpiCypher has begun offering SNAP-ChIP Certified Antibodies to widely studied histone PTMs, such as H3K4me3 and H3K27me3 (see Figure 1). Combined with SNAP-ChIP spike-in panels for antibody validation and data normalization, researchers now have unprecedented control over ChIP-seq experiments.
Which Antibody Should You Use in CUT&RUN?
CUT&RUN is a rapidly emerging technique in chromatin biology, generating high-resolution data with reduced cell inputs and sequencing requirements compared to ChIP-seq3-5. In CUT&RUN, immobilized cells are permeabilized and incubated with a factor-specific (e.g. H3K27me3) antibody. A fusion of Protein A / Protein G and micrococcal nuclease (pAG-MNase) is added to “tether” the MNase to antibody-bound loci. Following the addition of calcium, MNase cleaves adjacent chromatin, and these fragments diffuse out of the cell for streamlined collection and library preparation.
By avoiding bulk chromatin fragmentation and immunoprecipitation, CUT&RUN provides high quality data with low [signal : noise] and backgrounds vs. ChIP-seq. The use of pAG-MNase also enables accelerated sample processing. In fact, with EpiCypher’s most recent CUTANA™ CUT&RUN protocol, you can go from cells to sequencing data in less than 4 days!
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Defining H3K27me3 Antibody Performance in CUT&RUN
EpiCypher is taking a unique approach to addressing this problem, leveraging our SNAP-ChIP technology for in-depth comparisons of antibody performance in ChIP-seq vs. CUT&RUN. We recently applied this strategy to our SNAP-ChIP certified H3K4me3 antibody (read more). Here, we have performed a similar analysis of our recently released, SNAP-ChIP certified H3K27me3 antibody (Figure 1), demonstrating its reliable performance in CUT&RUN.
For this analysis, we did a head-to-head comparison of native ChIP-seq and CUT&RUN experiments performed using the same antibody. We examined genome tracks at representative loci (Figure 2) and also compared the genome-wide localization of H3K27me3 relative to transcription start sites (Figure 3).
As shown in Figure 2, the overall peak localization and amplitude is well conserved across ChIP-seq and CUT&RUN, and is also comparable to data sourced from ENCODE. Notably, the CUT&RUN genome tracks display lower background compared to all of the ChIP-seq datasets, despite the reduced sequencing depth used for this CUT&RUN experiment (8 million reads vs. 35-38 million for each ChIP-seq replicate), highlighting the improved resolution of immunotethering assays.
Previous studies have revealed peaks of H3K27me3 enrichment immediately following the transcription start site (TSS) of repressed genes, with intermediate levels in associated gene bodies8, 9. We examined enrichment of H3K27me3 at annotated human TSSs in our ChIP-seq and CUT&RUN datasets (Figure 3), and found patterns typical of H3K27me3 localization. CUT&RUN has noticeably lower background compared to ChIP, as indicated by a more defined drop-off between the blue (highly enriched) and white (non-enriched) regions in the heatmap (compare Figure 3A vs Figure 3B).
We also compared the genome-wide enrichment of H3K27me3 between our ChIP-seq and CUT&RUN datasets (Figure 4). This Pearson correlation analysis revealed a high degree of similarity between the two assays (r = 0.864), supporting that our SNAP-ChIP certified H3K27me3 antibody also performs reliably in CUT&RUN.
CUTANA™ CUT&RUN for Ultra-sensitive Chromatin Profiling
EpiCypher’s SNAP-ChIP certified H3K27me3 and H3K4me3 antibodies are both compatible with CUT&RUN assays, providing reliable tools for the most widely studied histone PTMs in chromatin literature. Combined with our CUTANA™ CUT&RUN accessory reagents and optimized protocol, this advance will allow scientists to profile diverse chromatin structures, including active (H3K4me3) and silenced (H3K27me3) regions, with confidence.
EpiCypher is currently developing CUTANA nucleosome spike-ins for directly testing antibody activity in CUT&RUN assays. Make sure to subscribe to our mailing list, or follow us on Twitter, to hear about our latest releases!
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References
1. Pfister SX, Ashworth A. Marked for death: targeting epigenetic changes in cancer. Nat Rev Drug Discov, 2017. 16(4): p. 241-63. (PubMed PMID: 28280262)
2. Berson A, et al. Epigenetic Regulation in Neurodegenerative Diseases. Trends Neurosci, 2018. 41(9): p. 587-98. (PubMed PMID: 29885742) (PMC6174532)
3. Skene PJ, Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife, 2017. 6: p. (PubMed PMID: 28079019) (PMC5310842)
4. Skene PJ, et al. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc, 2018. 13(5): p. 1006-19. (PubMed PMID: 29651053)
5. Meers MP, et al. Improved CUT&RUN chromatin profiling tools. Elife, 2019. 8: p. (PubMed PMID: 31232687)) (PMC6598765)
6. Shah RN, et al. Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies. Mol Cell, 2018. 72(1): p. 162-77 e7. (PubMed PMID: 30244833) (PMC6173622)
7.Grzybowski AT, et al. Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide. Mol Cell, 2015. 58(5): p. 886-99. (PubMed PMID: 26004229) (PMC4458216)
8.Wang Z, et al. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet, 2008. 40(7): p. 897-903. (PubMed PMID: 18552846)(PMC2769248)
9.Barski A, et al. High-resolution profiling of histone methylations in the human genome. Cell, 2007. 129(4): p. 823-37. (PubMed PMID: 17512414)