SNAP-ChIP® OncoStat™ Panel
Powered by BiozA panel of distinctly modified mononucleosomes assembled from recombinant human histones expressed in E. coli (two each of histones H2A, H2B, H3.3 and H4; accession numbers: H2A-P04908; H2B-O60814; H3.3-P84243*; H4-P62805) wrapped by 147 base pairs of barcoded Widom 601 positioning sequence DNA. The OncoStat panel is comprised of a pool of 1 unmodified (H3.3) plus 7 histone H3.3 point mutations: H3.3K4M, H3.3K9M, H3.3K27M, H3.3G34R, H3.3G34V, H3.3G34W, H3.3K36M. Each distinctly modified nucleosome is distinguishable by a unique sequence of DNA (“barcode”) at the 3’ end that can be deciphered by qPCR or next-generation sequencing. Each of the 8 nucleosomes in the pool is wrapped by 2 DNA species, each containing a distinct barcode (”A” and “B”, see SNAP-ChIP Manual) allowing for an internal technical replicate.
* Histone H3.2 contains a Cys to Ala mutation at position 110.
SNAP-ChIP OncoStat Panel
Schematic representation of nucleosomes comprising the OncoStat Panel: H3.3, H3.3K4M, H3.3K9M, H3.3K27M, H3.3G34R, H3.3G34V, H3.3G34W and H3.3K36M.
DNA Gel Data
Representative images for SNAP-ChIP OncoStats resolved by native PAGE and stained with ethidium bromide to visualize DNA. Lane 1: Free 147bp DNA used in nuclesome assembly (100 ng). Lane 2: Intact nucleosomes (200 ng).Comparable experiments were performed for the entire OncoStatPanel.
Protein Gel Data
Representative Coomassie stained PAGE gel of SNAP-ChIP OncoStats (2 μg each) to demonstrate the purity of the histones in the preparation. Sizes of molecular weight markers and positions of the core histones (H2A, H2B, H3.3 and H4) are indicated. Comparable experiments were performed for the entireOncoStat Panel.
ChIP Data
Representative chromatin imunoprecipitation (ChIP) data using commerically available antibodies targeting single point mutationsin histone H3.3. The antibodies were assayed in a native ChIP experiment with 3 μg antibody added to 3 μg HEK293 chromatin with theOncoStat Panel spiked-in prior to micrococcal nuclease digestion. Quantitative real-time PCR (qPCR) was used to measure recovery of duplicate DNA barcodes corresponding to each uniquely modified nucleosome in the panel (blue bars, X-axis). The black bars map to the log scale on the right y-axis and indicate the percentage of target immunoprecipitated relative to the input (a measure of the antibody efficiency). In each case, the SNAP-ChIP spike-in confirmed that the antibodies recovered the expected histone point mutation with high efficiency and specificity.
*Note: H3.3K4M was not assayed in the ChIP experiments due to a lack of commercially available antibodies.
Purified recombinant mononucleosomes, containing a mixture of 16 (1 unmodified plus 15 unique) H3 and H4 Purified recombinant mononucleosomes, containing a mixtureof 8 (1 unmodified plus 7 point mutations) nucleosomes in 10mM sodium cacodylate, pH 7.5, 100 mM NaCl, 1 mM EDTA,50% glycerol (w/v), 1x Protease Inhibitor cocktail, 100 ug/mLBSA, 10 mM β-mercaptoethanol. Molarity = 0.6 nM.MW = ~199283.7 Da (average MW of all 8 nucleosomes).
SNAP-ChIP OncoStats are highly purified recombinantmononucleosomes and are suitable for use as spike-in controlsfor ChIP reactions, for antibody specificity testing or for effectorprotein binding experiments. See manual for more information.
Product References:
SNAP-ChIP is adapted from Grzybowski et al (2015) Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide. Mol Cell 58: 886 – 899 LINK
