H3.3 Antibody, SNAP-Certified™ for CUT&RUN

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Figure 2: SNAP specificity analysis in CUT&RUN
CUT&RUN was performed as described in Figure 4. CUT&RUN sequencing reads were aligned to the unique DNA barcodes corresponding to each nucleosome in the variant panel (x-axis). Data are expressed as a percent relative to on-target recovery (H3.3 set to 100%).

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Figure 3: H3.3 CUT&RUN representative browser tracks
CUT&RUN was performed as described in Figure 4. Gene browser shots were generated using the Integrative Genomics Viewer (IGV, Broad Institute). Two representative loci of the top called peaks are shown. Similar results in peak structure and location were observed for both 500k and 50k cell inputs.

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Figure 4: CUT&RUN methods
CUT&RUN was performed on 500k and 50k K562 cells with the SNAP-CUTANA™ K-MetStat Panel (EpiCypher 19-1002) and Histone Variant Panel spiked-in prior to the addition of 0.5 µg of either IgG negative control (EpiCypher 13-0042), H3K4me3 positive control (EpiCypher 13-0060), or H3.3 antibodies. The experiment was performed using the CUTANA™ ChIC/CUT&RUN Kit v4 (EpiCypher 14-1048). Library preparation was performed with 5 ng of CUT&RUN enriched 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 5.2 million reads (IgG 500k cell input), 2.5 million reads (H3K4me3 500k cell input), 7.0 million reads (H3.3 500k cell input), and 3.7 million reads (H3.3 50k cell input). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.