Invitrogen™ OncoStat Panel, SNAP-ChIP™ Spike-in

Catalog No.	Quantity
A47355	100 reactions

Catalog No. A47355 Supplier Invitrogen™ Supplier No. A47355

Description

SNAP-ChIP (Sample Normalization and Antibody Profiling Chromatin ImmunoPrecipitation) uses DNA-barcoded semi-synthetic nucleosomes bearing distinct histone mutations as spike-in ChIP controls.

• SNAP-ChIP (Sample Normalization and Antibody Profiling Chromatin ImmunoPrecipitation) uses DNA-barcoded semi-synthetic nucleosomes bearing distinct histone mutations as spike-in ChIP controls.
• Homogenous, fully defined standards faithfully represent target mononucleosomes in the experimental sample
• Nucleosomes are subjected to rigorous quality control for lot-to-lot consistency
• Unique DNA barcodes can be distinguished from experimental sample genomes
• Spike-ins provide a direct readout of antibody performance in ChIP
• Ability to monitor technical variability between samples
• Defined standards enable universal normalization across experiments
• SNAP-ChIP panels are directly compatible with your current ChIP workflow, with semi-synthetic nucleosomes bearing the mutation of interest immunoprecipitated and processed alongside sample chromatin.
• Users can directly monitor antibody capability (specificity and enrichment) in a ChIP experiment by measuring the recovery of on- and off-target nucleosomes. The SNAP spike-ins can be further used as a defined standard to normalize your ChIP data, thereby controlling for technical variability across experiments.
• The OncoStat Panel consists of a pool of nucleosomes harboring seven well-studied histone H3.3 mutations that have been implicated in cancer (histone H3.3K4M, H3.3K9M, H3.3K27M, H3.3G34R, H3.3G34V, H3.3G34W and H3.3K36M) plus a wild-type H3.3 control. Further, each of these eight nucleosomes is wrapped by two independent DNA barcodes (16 total barcodes in the panel), allowing for an internal technical replicate in each ChIP reaction.
• A single spike-in of the panel allows users to check antibody specificity by examining the post-IP recovery of on- versus off-target SNAP-ChIP nucleosomes, supporting the generation of high quality ChIP data.

Specifications

• Description: Recombinant Nucleosome Panel
• Content And Storage: Store at -5 to -30°C.
• No. of Tests: 100 Reactions
• Quantity: 100 reactions
• Product Line: OncoStat, SNAP-ChIP
• Product Type: SNAP-ChIP Panel

Frequently Asked Questions (FAQs)

Can you please recommend specific histone PTM antibodies?

Thermo Fisher Scientific (https://www.thermofisher.com/us/en/home.html), in collaboration with EpiCypher, has performed extensive antibody testing for various histone PTMs, using SNAP-ChIP spike-in control panels to validate antibody specificity. For more information, please go to:
https://www.thermofisher.com/us/en/home/life-science/antibodies/invitrogen-antibody-validation/SNAP-ChIP-antibody-validation.html.

Will the SNAP-ChIP spike-ins affect the required sequencing depth?

The SNAP-ChIP spike-ins represent <<1% of total nucleosomes in the sample, so sequencing depth is unaffected.

With SNAP-ChIP panels, do you have specific guidelines for running the sequencing?

Paired-end sequencing is recommended for several reasons:
- Because the reverse barcodes are unique to each dNuc within a panel, they will not be reachable from the top strand in many NGS configurations (e.g., single end 50, 75, or 100 bp sequencing). Thus, half of the data associated with the SNAP-ChIP spike-ins (those from the top strand) cannot be confidently aligned to a specific nucleosome in the panel and will be discarded. The read depth associated with the SNAP-ChIP spike-ins will be concomitantly reduced.
- Paired-end sequencing allows read filtering to eliminate data associated with dinucleosomes (immunoprecipitated more efficiently than mononucleosomes* and thus overrepresented in the sequencing data). This bias can be mitigated by excluding fragments sized >220 bp from analysis.
* Grzybowski, A. T., Chen, Z. & Ruthenburg, A. J. Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide. Molecular Cell 58: 886-899, doi:10.1016/j.molcel.2015.04.022 (2015).

Can you please recommend a native ChIP protocol to use with SNAP-ChIP spike-in?

Please see the following reference for a detailed native ChIP method:
Brand, M., Rampalli, S., Chaturvedi, C. P. & Dilworth, F. J. Analysis of epigenetic modifications of chromatin at specific gene loci by native chromatin immunoprecipitation of nucleosomes isolated using hydroxyapatite chromatography. Nature Protocols 3: 398-409, doi:10.1038/nprot.2008.8 (2008).

I am planning to use SNAP-ChIP panels. Why should I use a native ChIP protocol with micrococcal nuclease (MNase) digestion vs. crosslinking/sonication?

SNAP-ChIP spike-in is directly compatible with both native and crosslinked approaches though the former is recommended. Crosslinking can impact antibody specificity and enrichment because crosslinked chromatin becomes more sticky and susceptible to epitope masking. In our experience, signal-to-noise ratios are often decreased in crosslinked samples compared to native ChIP.
In contrast, a native nuclei preparation that is micrococcal nuclease-digested to yield >95% pure mononucleosomes will yield samples that more closely resemble the SNAP-ChIP spike-ins (i.e., unfixed mononucleosomes). As a result, data obtained from the SNAP-ChIP controls will be most representative of the experimental samples.

With SNAP-ChIP panels, what results can I expect with the standard spike-in amount?

At a spike-in volume of 2 µL per 10 µg of chromatin, qPCR cycle threshold (Ct values) for the input samples is expected to be in the low ~20's. In a standard ChIP-sequencing experiment (30 million reads per sample), read counts for the individual SNAP-ChIP barcoded nucleosomes is expected to range from ~200-500. Based on these general expectations, users can make an educated decision about how much to scale the spike-in volume up or down in conditions where different amounts of sample chromatin are used.

Will SNAP-ChIP panel barcodes overlap with genomic DNA sequences from my samples?

SNAP-ChIP barcodes are designed to be compatible with multiple species (e.g., human, mouse, fly, and yeast) such that their genomic DNA can be readily distinguished without issue.

Why do I need SNAP-ChIP panels?

Think of SNAP-ChIP as a spike-in control: Both positive (modified nucleosomes with the PTM of interest) and negative (unmodified nucleosomes) controls are incorporated into a single spike-in. The standard method of performing qPCR for positive and negative genomic locus controls to evaluate performance of a ChIP experiment serves as an assumed proxy of on-target recovery, but does not directly assess recovery of the intended PTM. SNAP-ChIP spike-in provides the first ever direct and quantitative assessment of on-target recovery in every ChIP experiment. Additionally, the inclusion of related PTMs in one convenient spike-in panel gives information about antibody specificity, reducing concerns about the potential for undetected cross-reactivity to lead to incorrect assignment of biological function to a specific PTM (see Shah et al., Examining the Roles of H3K4 Methylation States With Systematically Characterized Antibodies. Molecular Cell 2018 Oct 4;72(1):162-177).

For Research Use Only. Not for use in diagnostic procedures.