SNAP-CUTANA™ HA Tag Panel

Panel of spike-in controls for CUT&RUN Assays

Product 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 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. 

Related Product

Pair with our HA Tag CUTANA™ CUT&RUN Antibody for CUT&RUN success.

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.

Storage and Stability

Store at -20°C. Lower temperatures can cause freezing and will permanently damage the magnetic beads. Stable for six months from date of receipt.

Validation Data

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.

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. 

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

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).

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.

Related Products

The SNAP-CUTANA K-MetStat Panel of spike-in controls is suitable for CUTANA CUT&RUN Assays.