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.

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.

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SNAP-CUTANA? HA Tag Panel

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