HA Tag CUTANA™ CUT&RUN Antibody

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Figure 2: HA-tagged transcription factor binding motif analysis in CUT&RUN
(A) Homer analysis determined that the GATA3 consensus motif, represented as a sequence logo position weight matrix, was enriched under GATA3-3xHA CUT&RUN peaks. (B) The number of GATA3-3xHA peaks containing GATA3 consensus motifs from panel A is represented by a Venn Diagram. (C-D) Two representative loci show overlap of GATA3-3xHA peaks with the consensus motifs noted by tick marks beneath the tracks.

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Figure 3: Western blot data
E. coli cells expressing a multi-tag fusion protein were used to prepare whole cell lysates. The indicated amounts (ng) of lysate were loaded onto a 4-20% SDS-PAGE gel and analyzed under standard western blot conditions using HA Tag antibody at a dilution of 1:25,000.

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Figure 4: Target-specific epitope cleavage of HA Tag antibody in CUT&RUN was determined using DNA-barcoded recombinant nucleosome spike-in controls
(A) A panel of recombinant nucleosomes was created where various epitope tags (3xTY1, 3xFLAG, 3xHA) were fused to the histone H3 tail. The fused nucleosomes and an unmodified control were immobilized to streptavidin beads (SA Bead) and spiked into CUT&RUN samples alongside ConA bead immobilized MDA-MB-231 cells expressing GATA3-3xHA (Figure 1). HA Tag antibody and pAG-MNase (EpiCypher 15-1016) were then added to release antibody-bound nucleosomes into solution through pAG-MNase mediated cleavage of the linker DNA (light blue). This approach provided a defined experimental control to assess whether the HA Tag antibody selectively cleaved the target epitope with high specificity and minimal background. (B) CUT&RUN sequence reads were aligned to the unique DNA "barcodes" corresponding to each nucleosome in the spike-in panel. Data are expressed as the percent of reads recovered relative to the intended target (3xHA, set to 100%). This analysis confirms that the HA Tag antibody specifically liberated the target epitope-tagged nucleosome into solution.

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Figure 5: CUT&RUN Methods
CUT&RUN was performed on 500k MDA-MB-231 cells stably expressing c-terminal 3xHA-tagged GATA3 [1]* with 0.5 µg of either HA Tag, H3K4me3 positive control (EpiCypher 13-0041), or IgG negative control (EpiCypher 13-0042) antibodies using the CUTANA™ ChIC/CUT&RUN Kit v2.0 (EpiCypher 14-1048). 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). Both kit protocols were adapted for high throughput Tecan liquid handling. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 2.4 million reads (IgG), 4.1 million reads (H3K4me3), and 8.6 million reads (HA Tag). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.