Histone H3K4me2 Antibody, SNAP-Certified™ for CUT&RUN

(Click to enlarge)

Figure 2: CUT&RUN genome wide enrichment
CUT&RUN was performed as described above. Sequence reads were aligned to 18,793 annotated transcription start sites (TSSs ± 2 kbp). Signal enrichment was sorted from highest to lowest (top to bottom) relative to the H3K4me2 - 50k cells sample (all gene rows aligned). High, medium, and low intensity are shown in red, yellow, and blue, respectively. H3K4me2 antibody produced the expected TSS enrichment pattern, which was consistent between 500k and 50k cells and greater than the IgG negative control.

(Click to enlarge)

Figure 3: H3K4me2 CUT&RUN representative browser tracks
CUT&RUN was performed as described above. Gene browser shots were generated using the Integrative Genomics Viewer (IGV, Broad Institute). Similar results in peak structure and location were observed for both cell inputs.

(Click to enlarge)

Figure 4: Antibody efficiency analysis in CUT&RUN using cell input correlation
CUT&RUN was performed as described above. Genome-wide correlation analysis was performed to compare H3K4me2 antibody enrichment using 500k cell and 50k cell inputs. The log of the number of reads per 75 bp binned region across the genome is plotted for both samples. CUT&RUN data generated using this H3K4me2 antibody are highly correlated between the two cell inputs (Pearson correlation r = 0.942), indicating high efficiency of H3K4me2 antibody target recovery.

(Click to enlarge)

Figure 5: Immunocytochemistry
ICC of HeLa cells using 2 µg/mL of H3K4me2 antibody (red). Actin filaments were labeled with fluorescein phalloidin (green).

(Click to enlarge)

Figure 6: Western blot data
Recombinant histone H3.3 (Lane 1) and acid extracts of HeLa cells (Lane 2) were blotted onto PVDF and probed with 0.025 µg/mL of H3K4me2 antibody.

(Click to enlarge)

CUT&RUN methods
CUT&RUN was performed on 500k and 50k K562 cells with the SNAP-CUTANA™ K-MetStat Panel (EpiCypher 19-1002) spiked-in prior to the addition of 0.5 µg of either H3K4me2 or IgG negative control (EpiCypher 13-0042) antibodies. The experiment was performed using the CUTANA™ ChIC/CUT&RUN Kit v2.0 (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 4.5 million reads (IgG 50k cell input), 7.2 million reads (H3K4me2 50k cell input) and 9.2 million reads (H3K4me2 500k 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.