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    EpiCypher新品推荐——SNAP-CUTANA™ DYKDDDDK Tag Panel

    2024-03-01
    浏览次数: 10

    SNAP-CUTANA™ DYKDDDDK Tag Panel是 CUT&RUN 的加标对照,提供了一种用于验证抗 FLAG®* 抗体的分析内对照,并证实了涉及FLAG表位标记的染色质蛋白的CUT&RUN反应的成功。这种重要的阳性对照可指导故障排除,以区分 FLAG 表位标记问题(包括转基因表达、标记蛋白的染色质结合、标签的溶剂可及性等)与 CUT&RUN 工作流程中的技术故障。该panel由两个包含未修饰组蛋白H3或3xDYKDDDDK-H3融合的核小体组成,每个核小体都包裹有两个独特的条形码DNA模板(A和B,用于内部技术复制)。核小体单独与顺磁珠偶联并汇集到单个panel中,方便一步加标到CUT&RUN反应。在添加抗 DYKDDDDK 或 IgG 阴性对照抗体之前,将 panel 与 ConA 固定的细胞一起添加(参见应用说明和表 1)。pAG-MNase释放基因组染色质和条形码核小体取决于所使用抗体的特异性。测序后,回收的 DYKDDDDK 与未修饰核小体的相对读取计数提供了靶向与脱靶回收率的定量指标(图 2),从而衡量实验成功率并指导故障排除工作。有关工作流集成、预期结果、数据分析和故障排除的详细信息,请参阅最新的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/)用户指南。


    *FLAG® is a registered trademark of Merck KGaA, Darmstadt, Germany and ANTI-FLAG is a trademark of Sigma-Aldrich Co. LLC.


    保存温度

    自收到之日起,-20℃下可稳定储存6个月。较低的温度会导致冻结,并会永久损坏磁珠。


    验证数据

    EpiCypher新品推荐——SNAP-CUTANA™ DYKDDDDK Tag Panel

    Figure 1: Schematic of SNAP-CUTANA™ DYKDDDDK Tag Panel

    The DYKDDDDK Tag Panel contains two nucleosomes - one has an H3 tail fusion to a 3xDYKDDDDK 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 14-104815-1016) during CUT&RUN. The nucleosomes are individually pre-conjugated to paramagnetic beads and pooled for convenient use.

    EpiCypher新品推荐——SNAP-CUTANA™ DYKDDDDK Tag Panel

    Figure 2: SNAP-CUTANA™ DYKDDDDK Tag Panel provides an in-assay control for CUT&RUN 

    reactions targeting FLAG-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™ DYKDDDDK Tag Panel. Data are expressed as a percent relative to on-target recovery (DYKDDDDK Tag set to 100%) or total counts (IgG). IgG antibody results demonstrate equal loading of unmodified and epitope nucleosomes in the panel. DYKDDDDK Tag antibody results show selective enrichment of the DYKDDDDK Tag spike-in nucleosomes, validating all CUT&RUN steps, including DYKDDDDK antibody binding, pAG-MNase cleavage, and wash conditions.

    EpiCypher新品推荐——SNAP-CUTANA™ DYKDDDDK Tag Panel

    Table 1: Recommended SNAP-CUTANA™ DYKDDDDK Tag Panel Spike-in dilution for CUT&RUN reactions of varying starting cell number.

    EpiCypher新品推荐——SNAP-CUTANA™ DYKDDDDK Tag Panel

    Figure 3: DNA gel data

    Nucleosomes in the SNAP-CUTANA™ DYKDDDDK 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).

    EpiCypher新品推荐——SNAP-CUTANA™ DYKDDDDK Tag Panel

    Figure 4: Protein gel data

    Coomassie stained SDS-PAGE gel of the nucleosome containing a 3xDYKDDDDK-H3 fusion (1 μg) in the SNAP-CUTANA DYKDDDDK Tag Panel demonstrates the purity of histones in the preparation. Sizes of molecular weight markers and positions of the core histones (H2A, H2B, 3xDYKDDDDK-H3, and H4) are indicated.

    EpiCypher新品推荐——SNAP-CUTANA™ DYKDDDDK Tag Panel

    Figure 5: CUT&RUN methods

    CUT&RUN was performed on 500k MDA-MB-231 native cells stably expressing 3xFLAG-tagged GATA3 [1]** using the CUTANA™ ChIC/CUT&RUN Kit v3 (EpiCypher 14-1048). SNAP-CUTANA™ DYKDDDDK Tag Panel was added just prior to the addition of either DYKDDDDK Tag (0.05 μg; EpiCypher 13-2031) 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.


    订购详情

    货号

    产品名称

    规格

    19-5001

    SNAP-CUTANA™ DYKDDDDK Tag Panel

    50 Reactions


    参考文献

    [1] Lowary & Widom J. Mol. Biol. (1998). PMID: 9514715

    [2] Takaku et al. Genome Biol. (2016). PMID: 26922637


     

    EpiCypher新品推荐——SNAP-CUTANA™ DYKDDDDK Tag Panel

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