SNAP-ChIP OncoStat Panel Description: A 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 mononucleosomes constitute a pool of 1 unmodified 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
* Histone H3.2 contains a Cys to Ala mutation at position 110.
SNAP-ChIP OncoStat Panel Formulation: 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 OncoStat Panel Storage and Stability: Stable for six months at -20°C from date of receipt.
SNAP-ChIP OncoStat Panel Application Notes: 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.
SNAP-ChIP is adapted from Grzybowski AT et al (2015) Mol Cell58: 886-889.
SNAP-ChIP OncoStat Panel Additional Resources and Files:
SNAP-ChIP OncoStat Manual
SNAP-ChIP OncoStat Full-length DNA Sequences - Useful for ChIP-seq data alignment
SNAP-ChIP OncoStat qPCR Primer Sequences - Formatted for easy upload for ordering from common vendors (e.g. IDT)
DNA Gel Data: Representative images for SNAP-ChIP OncoStatsresolved by native PAGE and stained with ethidium bromide tovisualize DNA. Lane 1: Free 147bp DNA used in nuclesomeassembly (100 ng). Lane 2: Intact nucleosomes (200 ng).Comparable experiments were performed for the entire OncoStatPanel. Email firstname.lastname@example.org for more info. (Click image to enlarge)
Protein Gel Data: Representative Coomassie stained PAGE gel ofSNAP-ChIP OncoStats (2 μg each) to demonstrate the purity of thehistones in the preparation. Sizes of molecular weight markers andpositions of the core histones (H2A, H2B, H3.3 and H4) areindicated. Comparable experiments were performed for the entireOncoStat Panel. Email email@example.com for more info. (Click image to enlarge)
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 ofduplicate DNA barcodes corresponding to each uniquely modified nucleosome in the panel (blue bars, X-axis). The black bars map to the logscale on the right y-axis and indicate the percentage of target immunoprecipitated relative to the input (a measure of the antibodyefficiency). In each case, the SNAP-ChIP spike-in confirmed that the antibodies recovered the expected histone point mutation with highefficiency and specificity. Email firstname.lastname@example.org for more info.
*Note: H3.3K4M was not assayed in the ChIP experiments due to a lack of commercially available antibodies. (Click image to enlarge)