SNAP-CUTANA?HA Tag Panel提供了用于驗證抗HA抗體和確認(rèn)涉及HA表位標(biāo)記的染色質(zhì)蛋白的成功CUT&RUN反應(yīng)的檢測控制。這一重要的陽性對照指導(dǎo)排除故障，以區(qū)分HA表位標(biāo)簽問題(包括轉(zhuǎn)基因表達、標(biāo)記蛋白的染色質(zhì)結(jié)合、標(biāo)簽的溶劑可及性等)與CUT&RUN工作流程中的技術(shù)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(nèi)部技術(shù)復(fù)制)。核小體分別與順磁珠偶聯(lián)，并匯集成一個Panel，方便一步插入到切割和運行反應(yīng)。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(xì)胞一起添加(見應(yīng)用說明和表1)。pAG-MNase釋放基因組染色質(zhì)和條形碼核小體取決于所用抗體的特異性。測序后，恢復(fù)的HA與未修飾的核小體的相對讀長計數(shù)提供了在靶恢復(fù)與脫靶恢復(fù)的定量指標(biāo)(圖2)，從而衡量實驗的成功率，并指導(dǎo)故障排除工作。有關(guān)工作流集成、預(yù)期結(jié)果、數(shù)據(jù)分析和故障排除的詳細(xì)信息，請參閱最新的CUTANA? CUT&RUN方法（https://www.epicypher.com/resources/protocols/cutana-cut-and-run-kit-manual）和SNAP-CUTANA? Spike-in（https://www.epicypher.com/resources/protocols/）用戶指南。
保存溫度
自收到之日起，-20℃下可穩(wěn)定儲存6個月。較低的溫度會導(dǎo)致凍結(jié)，并會永久損壞磁珠。
驗證數(shù)據(jù)
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.
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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
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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).
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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.
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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(chǎn)品名稱 | 規(guī)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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