SNAP-CUTANA™

SNAP-CUTANA™ HA Tag Panel

$295.00
SKU: 19-5002
Pack Size: 50 Reactions
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Description

The SNAP-CUTANA™ HA Tag Panel of spike-in controls for CUT&RUN offers an in-assay control to validate anti-HA antibodies and confirm the success of CUT&RUN reactions involving HA epitope-tagged chromatin proteins. This essential positive control guides troubleshooting to differentiate problems with HA epitope-tagging (including transgene expression, chromatin binding of the tagged protein, solvent accessibility of the tag, etc.) from technical failures in the CUT&RUN workflow. The panel consists of two nucleosomes containing unmodified histone H3 or 3xHA-H3 fusion, each wrapped with two uniquely barcoded DNA templates (A and B, for an internal technical replicate). The nucleosomes are individually conjugated to paramagnetic beads and pooled into a single panel for convenient one-step spike-in to CUT&RUN reactions. The panel is added alongside ConA-immobilized cells just prior to the addition of anti-HA or IgG negative control antibodies (see Application Notes and Table 1). The release of genomic chromatin and the barcoded nucleosomes by pAG-MNase is dependent on the specificity of the antibody used. After sequencing, the relative read count of recovered HA vs. unmodified nucleosomes provides a quantitative metric of on- vs. off-target recovery (Figure 2), thereby gauging experimental success and guiding troubleshooting efforts. See the most recent CUTANA™ CUT&RUN protocol and SNAP-CUTANA™ Spike-in User Guide for detailed information on workflow integration, expected results, data analysis, and troubleshooting.

Pair with our HA Tag CUTANA™ CUT&RUN Antibody for CUT&RUN success. Check it out here.

Validation Data

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Figure 1: Schematic of SNAP-CUTANA™ HA Tag Panel
The HA Tag Panel contains two nucleosomes - one has an H3 tail fusion to a 3xHA Tag epitope and one is an unmodified control. Both octamers are wrapped with two uniquely barcoded DNA templates (A and B). Each 250 bp DNA template contains a 123 bp 601 nucleosome positioning sequence (gray) [1], a unique 22 bp DNA-barcode (white; 4 barcodes total), and a 5’ biotin-TEG. The 5’ and 3’ linkers (blue) are compatible with cleavage by pAG-MNase (EpiCypher 14-1048, 15-1016) during CUT&RUN. The nucleosomes are individually pre-conjugated to paramagnetic beads and pooled for convenient use.

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Figure 2: SNAP-CUTANA™ HA Tag Panel provides an in-assay control for CUT&RUN reactions targeting HA-tagged proteins
CUT&RUN was performed as described in Figure 5. CUT&RUN sequencing reads were aligned to the unique DNA barcodes corresponding to each nucleosome in the SNAP-CUTANA™ HA Tag Panel. Data are expressed as a percent relative to on-target recovery (HA Tag set to 100%) or total counts (IgG). IgG antibody results demonstrate equal loading of unmodified and epitope nucleosomes in the panel. HA Tag antibody results show selective enrichment of the HA Tag spike-in nucleosomes, validating all CUT&RUN steps, including HA antibody binding, pAG-MNase cleavage, and wash conditions

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Table 1: Recommended SNAP-CUTANA™ HA Tag Panel Spike-in dilution for CUT&RUN reactions of varying starting cell number.

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Figure 3: DNA gel data
Nucleosomes in the SNAP-CUTANA™ HA Tag Panel were resolved via native PAGE and stained with ethidium bromide to confirm intact nucleosome assembly. Lane 1: Free 250 bp DNA used in nucleosome assembly (100 ng). Lane 2: Intact nucleosomes (400 ng).

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Figure 4: Protein gel data
Coomassie stained SDS-PAGE gel of the nucleosome containing a 3xHA-H3 fusion (1 μg) in the SNAP-CUTANA™ HA Tag Panel demonstrates the purity of histones in the preparation. Sizes of molecular weight markers and positions of the core histones (H2A, H2B, 3xHA-H3, and H4) are indicated.

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Figure 5: CUT&RUN methods
CUT&RUN was performed on 500k MDA-MB-231 native cells stably expressing 3xHA-tagged GATA3 [1]* using the CUTANA™ ChIC/CUT&RUN Kit v3 (EpiCypher 14-1048). SNAP-CUTANA™ HA Tag Panel was added just prior to the addition of either HA Tag (0.5 µg; EpiCypher 13-2010) or IgG negative control (0.5 µg; EpiCypher 13-0042) antibodies. Library preparation was performed with 5 ng of DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.

*Thanks to Dr. Takaku (UND) for 3xFLAG-GATA3-3xHA MDA-MB-231 cells.

Technical Information

Storage
Store at -20°C. Lower temperatures can cause freezing and will permanently damage the magnetic beads. Stable for six months from date of receipt.
To resuspend beads, gently mix into an even suspension by pipetting; DO NOT VORTEX
Formulation
A mixture of two semi-synthetic nucleosomes conjugated to paramagnetic beads in 10 mM sodium cacodylate pH 7.5, 100 mM NaCl, 1 mM EDTA, 50% glycerol (w/v), 1x Protease Inhibitor Cocktail, 100 μg/mL BSA, 10 mM β-mercaptoethanol.

Application Notes

See the most recent CUTANA™ CUT&RUN protocol and SNAP-CUTANA™ Spike-in User Guide for detailed information on workflow integration, expected results, data analysis, and troubleshooting. In Brief:

Product Use: Use the SNAP-CUTANA™ HA Tag Panel for reactions containing HA and IgG antibodies. Just before antibody addition in CUT&RUN, gently pipette to resuspend beads (do not vortex), then spike in 2 μL per 500k cells. If using less than the standard number of cells, decrease the amount of SNAP-CUTANA spike-in linearly by preparing a “working stock” dilution of the panel in Antibody Buffer, made fresh the day of use (Table 1). Adjust spike-in volume as needed aiming for the spike-in barcodes to comprise ~1% of the total unique sequencing reads. Table 1 gives recommended dilution amounts for varying numbers of starting cells, but optimization may be required for user-specific conditions.

Data Analysis: Detailed instructions are in the SNAP-CUTANA™ Spike-in User Guide (see Documents & Resources section below). Perform paired-end sequencing for a minimum of 50 bases. The Widom 601 DNA and DNA barcodes are distinct from human, mouse, fly, and yeast genomes such that they can be readily distinguished from sample chromatin. A shell script (.sh file extension) for spike-in alignment and an excel template for heatmap generation are available in the Documents & Resources section below. The shell script can be opened with any basic text editor program and contains detailed instructions hashed (#) at the beginning of the document. Make sure to copy and paste the R1 & R2 echo loop so there is a set for each reaction being analyzed.

References

Background References:
[1] Lowary & Widom J. Mol. Biol. (1998). PMID: 9514715
[2] Takaku et al. Genome Biol. (2016). PMID: 26922637
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