Histone H3K36me3 Antibody, SNAP-ChIP® Certified, CUTANA™ CUT&RUN Compatible *DISCONTINUED*

Histone H3K36me3 Antibody, SNAP-ChIP Certified, CUTANA™ Compatible References:

Grzybowski et al (2015) Mol Cell 58:886

Shah et al (2018) Mol Cell 72:162

Applications Key: ChIP-Chromatin IP; E-ELISA; FACS-Flow cytometry; IF-Immunofluorescence; IHC-Immunohistochemistry; ICC-Immunocytochemistry; L-Luminex; IP-Immunoprecipitation; WB-Western Blotting

Reactivity Key: B-Bovine; Ce-C. elegans; Ch-Chicken; Dm- Drosophila; Eu-Eukaryote; H-Human; M-Mouse; Ma-Mammal; R-Rat; Sc-S.cerevesiae; Sp-S. pombe; WR-Wide Range (predicted); X-Xenopus; Z-Zebrafish

Representative SNAP-ChIP-seq results: Cumulative histogram plot and heatmap of signal intensity depict H3K36me3 ChIP-seq data aligned to gene bodies (gene start - end, +/- 3.0 kb; left). Two representative genomic regions depicting H3K36me3 peak structure and distribution are shown (right). Data shown are representative of H3K36me3 ChIP antibody (EpiCypher Catalog No. 13-0031) and are not lot-specific. Native ChIP-seq was performed using K562 cells as described (Shah et al., Mol Cell 2018) with SNAP-ChIP^TM K-MetStat Spike-in (Catalog No. 19-1001) nucleosome controls added prior to chromatin digestion to confirm antibody specificity and ChIP efficiency. Paired-end sequencing libraries were prepared using the NEBNext^® Ultra^TM II DNA Library Prep Kit for Illumina^®. ChIP libraries were sequenced on an Illumina^® NextSeq. Sequencing reads were aligned to the human genome using Bowtie 2 (Johns Hopkins University). Bigwig files of read enrichment in binned genomic regions (signal intensity) flanking the indicated gene features were used to create a cumulative histogram plot and heatmap of signal intensity ( www.basepairtech.com). Gene browser shots were generated using the Integrative Genomics Viewer (IGV, Broad Institute) with the window size denoted (top).
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Luminex Mutliplexed Specificity Profiling: Histone H3K36me3 antibody was assessed using a Luminex® based approach employing dCypher® Nucleosome K-MetStat Panel (EpiCypher Catalog No. 16-9002). The panel comprises biotinylated designer nucleosomes (x-axis) individually coupled to color coded Luminex Magplex® beads. Antibody binding to the panel of 16 nucleosomes was tested in multiplex at a 1:1000 dilution, and detected with second layer anti-IgG*PE. Data was generated using a Luminex FlexMAP3D®. Data normalized to relevant on-target (H3K36me3; set to 100)
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SNAP-ChIP Data: Histone H3K36me3 antibody (5 µg) was tested in a native ChIP experiment using chromatin from K-562 cells (5 µg) with the SNAP-ChIP K-MetStat Panel (EpiCypher Catalog No. 19-1001) spiked-in prior to micrococcal nuclease digestion. Specificity (left y-axis) was determined by qPCR for the DNA barcodes corresponding to modified nucleosomes in the SNAP-ChIP panel (x-axis). Black bar represents antibody efficiency (right y-axis; log scale) and indicates percentage of the target immunoprecipitated relative to input. Error bars represent mean ± SEM in replicate ChIP experiments.
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H3K36me3 SNAP-ChIP-seq and CUT&RUN representative tracks: A gene browser shot generated using the Integrative Genomics Viewer (IGV, Broad Institute) shows a representative locus for EpiCypher H3K36me3 ChIP-seq (blue tracks, 3 μg antibody) and CUT&RUN (green track, 1:100 antibody dilution). For comparison ENCODE H3K36me3 ChIP-seq using a different antibody is shown (bottom orange track, GEO accession number GSM621387). Similar results in peak structure and location were observed throughout the genome for EpiCypher H3K36me3 antibody in ChIP-seq and CUT&RUN. Methods: Native ChIP-seq was performed as described (Shah et al., Mol Cell 2018). CUT&RUN was performed using EpiCypher CUTANA pAG-MNase for ChIC/CUT&RUN (EpiCypher Catalog No. 15-1016) as described (EpiCypher.com/cutana-protocol). Library preparation was performed with 10 ng DNA using the NEBNext^® Ultra^TM II DNA Library Prep Kit for Illumina^®. ChIP libraries were sequenced on an Illumina NextSeq 550 (2x150bp paired end). The total number of reads was 33.6 million for ChIP-seq and 3.2 million for CUT&RUN.
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ChIP-seq and CUT&RUN genome wide analysis EpiCypher H3K36me3 antibody was tested in native ChIP-seq (A) and CUT&RUN (B) using the methods described above. Genome-wide analysis of H3K36me3 enrichment (signal intensity) flanking annotated genes (gene start to gene end; +/- 3kb) is graphed as a cumulative histogram plot (top) and shown in a heatmap (bottom). Individual gene loci in each row of the heatmap are colored by signal intensity and sorted by strongest to lowest enrichment (top to bottom). EpiCypher H3K36me3 antibody displays a characteristic enrichment pattern in gene bodies.
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ChIP-seq vs. CUT&RUN correlation analysis Genome-wide correlation analysis was performed to compare EpiCypher H3K36me3 antibody enrichment in ChIP-seq and CUT&RUN. The number of reads per 5 kb binned region across the genome is plotted for CUT&RUN (x-axis) vs. ChIP-seq (y-axis) (EaSeq). ChIP-seq and CUT&RUN data generated using this antibody are highly correlated (Pearson correlation r = 0.786).
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Western blot analysis Recombinant histone H3.3 (Lane 1) and acid extracts of HeLa cells (Lane 2) were blotted onto PVDF and probed with 2 μg/mL Histone H3K36me3 antibody.
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