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.

19-5002

SNAP-CUTANA™   HA Tag Panel

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