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

The SNAP-ChIP spike-ins represent Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center .

Q: 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). Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center .

Question 1

Can you please recommend specific histone PTM antibodies?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 2

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

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 3

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

Accepted Answer

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).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 4

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

Accepted Answer

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).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 5

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

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 6

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

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 7

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

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 8

Why do I need SNAP-ChIP panels?

Accepted Answer

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).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

K-AcylStat Panel, SNAP-ChIP™ Spike-in, 10 Reactions - FAQs