SNAP-CUTANA™HA Tag Panel提供了用于驗證抗HA抗體和確認涉及HA表位標記的染色質蛋白的成功CUT&RUN反應的檢測控制。這一重要的陽性對照指導排除故障，以區分HA表位標簽問題(包括轉基因表達、標記蛋白的染色質結合、標簽的溶劑可及性等)與CUT&RUN工作流程中的技術故障。該面板由兩個包含未修飾組蛋白H3或3xHA-H3融合的核小體組成，每個核小體都包裹有兩個獨-特的條形碼DNA模板(A和B，用于內部技術復制)。核小體分別與順磁珠偶聯，并匯集成一個Panel，方便一步插入到切割和運行反應。在添加抗ha或IgG陰性對照抗體之前，將該檢測組合與ConA固定的細胞一起添加(見應用說明和表1)。pAG-MNase釋放基因組染色質和條形碼核小體取決于所用抗體的特異性。測序后，恢復的HA與未修飾的核小體的相對讀長計數提供了在靶恢復與脫靶恢復的定量指標(圖2)，從而衡量實驗的成功率，并指導故障排除工作。有關工作流集成、預期結果、數據分析和故障排除的詳細信息，請參閱最新的CUTANA™ CUT&RUN方法（相關鏈接可找Epicypher代理商欣博盛生物獲取）和SNAP-CUTANA™ Spike-in（相關鏈接可找Epicypher代理商欣博盛生物獲取）用戶指南。
保存溫度
自收到之日起，-20℃下可穩定儲存6個月。較低的溫度會導致凍結，并會永-久損壞磁珠。
驗證數據
Figure 1: Schematic of SNAP-CUTANA™ HA Tag Panel |
The HA Tag Panel contains two nucleosomes - one has an H3 tail fusion to a 3xHA Tag epitope and one is an unmodified control. Both octamers are wrapped with two uniquely barcoded DNA templates (A and B). Each 250 bp DNA template contains a 123 bp 601 nucleosome positioning sequence (gray) [1], a unique 22 bp DNA-barcode (white; 4 barcodes total), and a 5’ biotin-TEG. The 5’ and 3’ linkers (blue) are compatible with cleavage by pAG-MNase (EpiCypher 14-1048, 15-1016) during CUT&RUN. The nucleosomes are individually pre-conjugated to paramagnetic beads and pooled for convenient use. |
Figure 2: SNAP-CUTANA™ HA Tag Panel provides an in-assay control for CUT&RUN reactions targeting HA-tagged proteins |
CUT&RUN was performed as described in Figure 5. CUT&RUN sequencing reads were aligned to the unique DNA barcodes corresponding to each nucleosome in the SNAP-CUTANA™ HA Tag Panel. Data are expressed as a percent relative to on-target recovery (HA Tag set to 100%) or total counts (IgG). IgG antibody results demonstrate equal loading of unmodified and epitope nucleosomes in the panel. HA Tag antibody results show selective enrichment of the HA Tag spike-in nucleosomes, validating all CUT&RUN steps, including HA antibody binding, pAG-MNase cleavage, and wash conditions |
Table 1: Recommended SNAP-CUTANA™ HA Tag Panel Spike-in dilution for CUT&RUN reactions of varying starting cell number. |
Figure 3: DNA gel data |
Nucleosomes in the SNAP-CUTANA™ HA Tag Panel were resolved via native PAGE and stained with ethidium bromide to confirm intact nucleosome assembly. Lane 1: Free 250 bp DNA used in nucleosome assembly (100 ng). Lane 2: Intact nucleosomes (400 ng). |
Figure 4: Protein gel data |
Coomassie stained SDS-PAGE gel of the nucleosome containing a 3xHA-H3 fusion (1 μg) in the SNAP-CUTANA™ HA Tag Panel demonstrates the purity of histones in the preparation. Sizes of molecular weight markers and positions of the core histones (H2A, H2B, 3xHA-H3, and H4) are indicated. |
Figure 5: CUT&RUN methods |
CUT&RUN was performed on 500k MDA-MB-231 native cells stably expressing 3xHA-tagged GATA3 [1]* using the CUTANA™ ChIC/CUT&RUN Kit v3 (EpiCypher 14-1048). SNAP-CUTANA™ HA Tag Panel was added just prior to the addition of either HA Tag (0.5 µg; EpiCypher 13-2010) or IgG negative control (0.5 µg; EpiCypher 13-0042) antibodies. Library preparation was performed with 5 ng of DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions. *Thanks to Dr. Takaku (UND) for 3xFLAG-GATA3-3xHA MDA-MB-231 cells. |
訂購詳情
貨號 | 產品名稱 | 規格 |
19-5002 | SNAP-CUTANA™ HA Tag Panel | 50 Reactions |
參考文獻
[1] Lowary & Widom J. Mol. Biol. (1998). PMID: 9514715
[2] Takaku et al. Genome Biol. (2016). PMID: 26922637
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