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EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

2024-03-01
浏览次数: 23

H3K27ac(组蛋白H3赖氨酸27乙酰化)抗体符合EpiCypher的批次特异性SNAP-Certified™标准,在CUT&RUN和CUT&Tag应用中具有特异性和高效的靶标富集。这需要与相关组蛋白 PTMs (使用加标对照的 SNAP-CUTANA™ K-AcylStat Panel 测定,EpiCypher RD193002)的交叉反应性 <20%(图 1 和 图5)。在不同的细胞起始条件下,一致的基因组富集结果证实了高靶标效率:CUT&RUN中500k和50k的细胞量(图2-3),CUT&Tag中100k和10k的细胞量(图6-7)。即使在细胞数量减少的情况下,高效抗体也显示出相似的峰结构(图3和7)和高度保守的全基因组信号(图2和6)。H3K27ac与基因激活相关,并在活性增强子和启动子中富集[1]。


产品详情

产品名称:H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

宿主来源:Rabbit

实验应用:CUT&RUN, CUT&Tag

免疫原:A synthetic peptide corresponding to histone H3 acetylated at lysine 27

克隆性:Monoclonal[2114-3E4]

保存温度:自收到之日起,4℃下可稳定储存1年。


验证数据

—— CUT&RUN


EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

Figure 1: Average SNAP specificity analysis from two CUT&RUN experiments

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-AcylStat panel (x-axis). Data are expressed as a percent relative to on-target recovery (H3K27ac set to 100%). The antibody showed recovery of H3K27ac spike-in nucleosomes as well as H3K27ac nucleosomes that contain a proximal phosphorylation at S28 at both 500k and 50k cells. The antibody cross-reacts with extended acyl states (butyrylation and crotonylation) at H3K27, but these are typically low abundance in cells [2].

EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

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 H3K27ac - 500k cells reaction (all gene rows aligned). High, medium, and low intensity are shown in red, yellow, and blue, respectively. H3K4me3 positive control and H3K27ac antibodies produced the expected enrichment pattern, which was consistent between 500k and 50k cells and greater than the IgG negative control.

EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

Figure 3: H3K27ac 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). H3K27ac antibody tracks display peaks at promoters and enhancers, consistent with the biological function of this PTM. Similar results in peak structure and locations were observed for both 500k and 50k cell inputs.

EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

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) or SNAP-CUTANA™ K-AcylStat Panel (EpiCypher RD193002) spiked-in prior to the addition of 0.5 µg of either IgG negative control (EpiCypher 13-0042), H3K4me3 positive control (EpiCypher 13-0041), or H3K27ac 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 3.8 million reads (IgG 500k cell input), 5.0 million reads (IgG 50k cell input), 2.5 million reads (H3K4me3 500k cell input), 9.1 million reads (H3K4me3 50k cell input), 12.6 million reads (H3K27ac 500k cell input), and 11.0 million reads (H3K27ac 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.


—— CUT&Tag

EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and 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-AcylStat panel (x-axis). Data are expressed as a percent relative to on-target recovery (H3K27ac set to 100%). The antibody showed recovery of H3K27ac spike-in nucleosomes and to a lesser extent H3K27ac nucleosomes that contain a proximal phosphorylation at S28 at both 500k and 50k cells. The antibody cross-reacts with butyrylation at H3K27, but this is typically low abundance in cells [2].


EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

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 H3K27ac - 100k nuclei reaction (all gene rows aligned). High, medium, and low intensity are shown in red, yellow, and blue, respectively. H3K4me3 positive control and H3K27ac antibodies produced the expected enrichment pattern, which was consistent between 100k and 10k nuclei and greater than the IgG negative control.



EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

Figure 7: H3K27ac 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). H3K27ac antibody tracks display peaks at promoters and enhancers, consistent with the biological function of this PTM. Similar results in peak structure and locations were observed for both 100k and 10k nuclei inputs.


EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

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) or SNAP-CUTANA™ K-AcylStat Panel (EpiCypher RD193002) spiked-in prior to the addition of 0.5 µg of either IgG negative control (EpiCypher 13-0042), H3K4me3 positive control (EpiCypher 13-0041), or H3K27ac 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), 3.5 million reads (H3K4me3 100k nuclei input), 8.0 million reads (H3K4me3 10k nuclei input), 8.1 million reads (H3K27ac 100k nuclei input) and 8.5 million reads (H3K27ac 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.

 

订购详情

货号

产品名称

规格

13-0059

H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

100 µg


参考文献

[1] Pei et al. Clinical Epigenetics (2020). PMID: 32664951

[2] Simithy et al. Nature Communications (2017). PMID: 29070843

 


EpiCypher新品推荐——H3K27ac Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag

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