HA Tag CUTANA™ CUT&RUN Antibody

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Figure 2: HA-tagged protein peaks in CUT&RUN
Heatmaps show HA Tag antibody-enriched peaks called for GATA3-3xHA overexpressing cells (OE) in aligned rows relative to all other experimental conditions. Rows are ranked by intensity (top to bottom) and colored such that red indicates high localized enrichment and blue denotes background signal. A high degree of overlap is observed for HA and GATA3 antibodies as expected in the OE cells, while empty vector control (Ctrl) shows absence of HA and lower GATA3 enrichment representing endogenous protein. IgG shows low background signal. H3K4me3, a canonical mark of promoters, does not appear in regions of high GATA3 enrichment.

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Figure 3: CUT&RUN representative browser tracks for HA-tagged protein
Gene browser shows were generated using the Integrative Genomics Viewer (IGV, Broad Institute). Two of the top called peaks in GATA3-3xHA overexpressing (OE) cells are shown. The peaks show the same distribution patterns as observed in the genome-wide heatmaps (Figure 2). IgG shows low background, H3K4me3 is unchanged between empty vector control (Ctrl) and OE cells, GATA3 peaks are more robust in OE cells, and peaks overlap between GATA3 and HA antibodies in OE cells. These results demonstrate the robustness and specificity of HA Tag antibody in CUT&RUN experiments targeting HA-tagged proteins.

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Figure 4: 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 5: CUT&RUN Methods
CUT&RUN was performed on 500k MDA-MB-231 native cells stably overexpressing c-terminal 3xHA-tagged GATA3 [1] or containing vector control. 0.5 µg of either HA Tag, H3K4me3 (EpiCypher 13-0060), GATA3 (CST 5852), or IgG (EpiCypher 13-0042) antibodies were used with the CUTANA™ ChIC/CUT&RUN Kit v4 (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™ Library Prep Kit (EpiCypher 14-1001/14-1002). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth (vector control cells/overexpressing cells) was 5.9/6.2 million reads (IgG), 13.7/12.6 million reads (H3K4me3), 8.1/5.2 million reads (GATA3), and 5.7/5.9 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.