H3K4me3

H3K4me3 Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

$525.00
SKU: 13-0060
Pack Size: 100 µg
  • Type:  Monoclonal [2909-3D7]
  • Host:  Rabbit
  • Applications:  CUT&RUN, CUT&Tag
  • Reactivity:  Human, Wide Range (Predicted)
  • Format:  Antigen affinity-purified
  • Target Size:  15 kDa

Description

This H3K4me3 (histone H3 trimethylated at lysine 4) antibody meets EpiCypher’s lot-specific SNAP-Certified™ criteria for specificity and efficient target enrichment in both CUT&RUN and CUT&Tag applications. This requires <20% cross-reactivity to related histone PTMs determined using the SNAP-CUTANA™ K-MetStat Panel of spike-in controls (EpiCypher 19-1002, Figures 1 and 5). High target efficiency is confirmed by consistent genomic enrichment at varying cell inputs: 500k and 50k cells in CUT&RUN (Figures 2-3); 100k and 10k cells in CUT&Tag (Figures 6-7). High efficiency antibodies display similar peak structures (Figures 3 and 7) and highly conserved genome-wide signal (Figures 2 and 6) even at reduced cell numbers. This antibody targets histone H3K4me3, which is enriched at active promoters near transcription start sites (TSS) and promotes gene activation.

Validation Data - CUT&RUN

Figure 1: 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 K-MetStat panel (x-axis). Data are expressed as a percent relative to on-target recovery (H3K4me3 set to 100%). The antibody showed highly specific recovery of H3K4me3 spike-in nucleosomes at both 500k and 50k cells.

Figure 2: CUT&RUN genome-wide enrichment
CUT&RUN was performed as described in Figure 4. 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 H3K4me3 - 500k cells reaction (all gene rows aligned). High, medium, and low intensity are shown in red, yellow, and blue, respectively. H3K4me3 antibodies produced the expected enrichment pattern, which was consistent between 500k and 50k cells and greater than the IgG negative control.

Figure 3: H3K4me3 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). H3K4me3 antibody tracks display sharp peaks at gene promoters, consistent with the biological function of this PTM. Similar results in peak structure and location were observed for both 500k and 50k cell inputs.

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) spiked-in prior to the addition of 0.5 µg of either IgG negative control (EpiCypher 13-0042) or H3K4me3 antibodies. The experiment was performed using the CUTANA™ ChIC/CUT&RUN Kit v3 (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 3.5 million reads (IgG 500k cell input), 4.0 million reads (IgG 50k cell input), 5.6 million reads (H3K4me3 500k cell input), and 2.8 million reads (H3K4me3 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.

Validation Data - CUT&Tag

Figure 5: SNAP specificity analysis in CUT&Tag
CUT&Tag was performed as described in Figure 8. CUT&Tag sequencing reads were aligned to the unique DNA barcodes corresponding to each nucleosome in the K-MetStat panel (x-axis). Data are expressed as a percent relative to on-target recovery (H3K4me3 set to 100%). The antibody showed highly specific recovery of H3K4me3 spike-in nucleosomes at both 100k and 10k nuclei.

Figure 6: CUT&Tag genome-wide enrichment
CUT&Tag was performed as described in Figure 8. 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 H3K4me3 - 100k nuclei reaction (all gene rows aligned). High, medium, and low intensity are shown in red, yellow, and blue, respectively. H3K4me3 antibodies produced the expected enrichment pattern, which was consistent between 100k and 10k nuclei and greater than the IgG negative control.

Figure 7: H3K4me3 CUT&Tag representative browser tracks
CUT&Tag was performed as described in Figure 8. Gene browser shots were generated using the Integrative Genomics Viewer (IGV, Broad Institute). H3K4me3 antibody tracks display sharp peaks at gene promoters, consistent with the biological function of this PTM. Similar results in peak structure and location were observed for both 100k and 10k nuclei inputs.

Figure 8: CUT&Tag methods
CUT&Tag was performed on 100k and 10k K562 nuclei with the SNAP-CUTANA™ K-MetStat Panel (EpiCypher 19-1002) spiked-in prior to the addition of 0.5 µg of either IgG negative control (EpiCypher 13-0042) or H3K4me3 antibodies. The experiment was performed using the CUTANA™ CUT&Tag Kit v1 (EpiCypher 14-1102/14-1103). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 1.3 million reads (IgG 100k nuclei input), 2.0 million reads (IgG 10k nuclei input), 6.9 million reads (H3K4me3 100k nuclei input), and 10.6 million reads (H3K4me3 10k nuclei 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.

Technical Information

Immunogen
A synthetic peptide corresponding to histone H3 trimethylated at lysine 4
Storage
Stable for 1 year at 4ºC from date of receipt
Formulation
Antigen affinity-purified recombinant monoclonal antibody in borate buffered saline pH 8.0, 0.09% sodium azide

Recommended Dilution

CUT&RUN
0.5 µg per reaction
CUT&Tag
0.5 µg per reaction

Gene & Protein Information

Uniprot ID
H3.1 - P68431
Alternate Names
H3, H3/a, H3/b, H3/d

Documents & Resources

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