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EpiCypher新品促销—— CUTANA™ CUT&Tag Kit升级版（V 2）

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EpiCypher新品促销—— CUTANA™ CUT&Tag Kit升级版（V 2）

EpiCypher新品促销—— CUTANA™ CUT&Tag Kit升级版（V 2）


EpiCypher CUTANA™ CUT&Tag Kit升级版（Version 2）来了！升级版各方面表现都优于V1版本，

助您持续生成高质量的CUT&Tag数据。新品推广期间凡订购EpiCypher CUTANA™ CUT&Tag Kit，可享额外20%折扣优惠！

前30名下单的客户，还有好礼相送（加油鸭挂件一个）。

 

促销时间：即日起-2024.9.25

促销代码：CTK20

 

【 促销产品清单 】

供应商

产品货号

产品名称

产品规格

EpiCypher     

14-1102       

CUTANA™ CUT&Tag Kit with Primer Set 1      

48 Reactions      

EpiCypher

14-1103

CUTANA™ CUT&Tag Kit with Primer Set 2

48 Reactions

【 六个常见问题带您了解CUT&Tag新变革 】

EpiCypher新品促销—— CUTANA™ CUT&Tag Kit升级版（V 2）

从单细胞图谱分析、空间表观基因组学到 FFPE 样本分析，CUT&Tag正在成为研究热点1-7。 CUT&Tag也是EpiCypher的研发重点。多年来，EpiCypher的团队开发并优化了多种表观基因组图谱检测方法，包括CUT&Tag、CUT&RUN和ChIP-seq。EpiCypher团队充分利用专业知识研发的CUTANA™ CUT&Tag试剂盒，使这项令人难以置信的技术能够走进全球更多的研究领域和实验室。CUTANA™ CUT&Tag 试剂盒第2版现已上市，而且各方面都优于之前的版本。

 

从表面上看，CUT&Tag非常简单8,9。Protein A/G的融合体在抗体标记的染色质上栓了一种过度活跃的Tn5转座酶。激活的Tn5插入接头序列并切割DNA，这一过程被称为标签化。在EpiCypher的Direct-to-PCR方法中，使用识别接头序列的引物从反应混合物中直接扩增标记DNA，绕过文库制备，提高对低细胞数的敏感性。整个测定过程使用的是与磁珠结合的完整细胞核，并对在使用8联排管时的方法进行了优化，从而实现高通量工作流程和自动化解决方案。

 

为了简化实验过程，EpiCypher团队开发了CUTANA™ CUT&Tag试剂盒，其中包括从细胞到纯化的测序文库所需的所有试剂。请注意，CUT&Tag仅推荐用于组蛋白翻译后修饰（PTMs）的定位。 欲进一步了解如何使用CUT&Tag与其姊妹技术CUT&RUN，请您参阅这篇文章：http://www.jinpanbio.com/xwzx_2376.html。

 

尽管已取得了一些进展，但CUT&Tag仍然是一项具有挑战性的表观基因组检测技术。为了使其方法更加可靠，EpiCypher研发团队不断测试CUTANA™ CUT&Tag实验方案，研究不同的缓冲液成分，调整反应条件，并测试了多种细胞类型。CUTANA™ CUT&Tag试剂盒的第二版展现了EpiCypher最新的优化成果。

 

✍ 为什么科研学者会对CUT&Tag感兴趣？

科研人员之所以对CUT&Tag非常感兴趣，是因为它可以让科学家在不降低数据质量的情况下简化实验流程，这是ChIP不可能做到的。自从CUT&Tag出现以来，研发人员终于能够开发出更高通量的染色质图谱分析。与ChIP-seq相比，CUT&Tag可以使用更少的细胞，花费更少的时间，对更多的反应进行多重测序，并生成更高分辨率的数据。

 

✍ 为什么CUT&Tag比ChIP-seq和CUT&RUN快得多呢？

由于各种原因，CUT&Tag比其他染色质图谱分析技术更快。从样本处理的角度看，CUT&Tag无需再多花时间优化细胞裂解、交联或染色质碎裂。CUT&Tag使用完整的细胞核，可在15分钟内从新鲜或冷冻的细胞中获取。然后将细胞核与固体支撑物（ConA磁珠）结合，这样就能限制样品的损失，并可使用磁力架快速清洗。这一特点使该方法与8联排管兼容，进一步加快了实验速度。

从实验方法的角度看，直接插入测序接头无需进行标准的文库准备步骤，不仅节省了一天的工作时间，而且减少了样品损失。大多数CUT&Tag实验流程中需要纯化DNA，然后进行 PCR，而EpiCypher的Direct-to-PCR策略允许实验人员直接从CUT&Tag反应中扩增标记的DNA。因为所有的实验过程都是在8联排管中进行的，所以实验人员可以将接头引物和PCR混合物直接加入到CUT&Tag反应管中。对于时间紧张的项目，实验人员可以在CUT&Tag第2天结束时加载测序仪，第3天就可以开始处理数据。对于科研人员来讲，这样的周转时间非常宝贵。

 

✍ 与ChIP-seq相比，为什么在CUT&Tag中可以使用更少的细胞呢?

因为简化了实验流程，降低了背景信号影响，所以CUT&Tag需要的起始细胞数量较少。在 ChIP-seq中，需要交联和染色质超声处理或片段化来制备input的染色质。在免疫沉淀（IP）步骤中，抗体被添加到染色质片段化池中。理想情况下，抗体只与靶标结合，但免疫沉淀几乎总是能回收非靶标片段，并在测序数据中引入背景信号或测序假象。科学家通常会增加细胞数量以克服背景信号影响并提高数据质量，但这种解决方法会限制低数量细胞的应用。

 

CUT&Tag使用与磁珠结合的完整细胞核，不需要按照传统的IP步骤进行实验，从而减少了背景信号影响。EpiCypher的方法还绕过了ChIP和标准文库制备中的多个DNA纯化步骤，有助于减少样本的损失。总之，与ChIP-seq相比，CUT&Tag可以减少约10倍的材料，这是非常不可思议的。

 

✍ 科研学者在使用CUT&Tag实验操作流程时最常见的问题是什么呢?

研究者进行CUT&Tag实验时遇到的主要问题是产量低甚至没有产量，这可能是由多种变量影响的。产率低通常是由样品预处理不佳、实验过程中样品损失、ConA磁珠结合不上以及反应混合问题造成的。这些问题都有关联，因此解决起来比较复杂。

 

样品制备不良的表现是细胞裂解、细胞核制备中有碎片或ConA磁珠结合后上清液中存在未结合的细胞核。如果样本预处理出现上述情况，就可能无法进行标签化，进而导致产量低。

 

混合不充分也是产量低的一个常见原因。保持磁珠在溶液中对检测的成功至关重要：它有助于确保抗体和pAG-Tn5的均匀分布，并有助于高效的indexing PCR。然而，在实验方案中的第2天，ConA 磁珠浆会变得粘稠且难以重悬，尤其是在标记之后。虽然在处理材料时应当温和，但如果不能很好的混合CUT&Tag反应物，就会严重降低产量。 EpiCypher实验方案详细说明了何时以及如何混合样品，以便最终稳定地回收CUT&Tag生成的文库。

 

✍ 新版CUT&Tag试剂盒有什么亮点呢?

版本更新的重点是帮助使用者持续生成高质量的CUT&Tag数据。EpiCypher的团队详细讨论了实验流程的每个步骤。比如，为什么缓冲液要使用某种成分或pH值？对细胞核或细胞生理有何影响？这些问题帮助EpiCypher团队找到了可以改进的关键点。

 

pAG-Tn5的表征表明，酶本身并不是影响产量问题所在，且标记反应是高效的，但样品在标记后的实验步骤中损失了。为此，EpiCypher团队对样品处理、缓冲液成分、方案步骤进行了广泛的头对头比较研究，并对优秀的竞争产品进行了测试。

 

这些实验揭开了问题的神秘面纱。TAPS Buffer中的低盐浓度导致了渗透性变化和细胞核裂解。混合技术也尤为重要，它是造成样品损失的原因之一。例如，加入SDS Release Buffer后，样品变得粘稠，无法移液。在实验方案的其他部分，由于涡旋使材料粘在离心管边上，也会导致样品损失。

 

根据实验结果，EpiCypher团队去掉了标记后的低盐 TAPS Buffer洗涤，取而代之的是含有生理盐的Pre-Wash Buffer，以保持细胞核的完整性。EpiCypher团队还完善了实验流程中具体的重悬浮和混合方法，以帮助指导使用者进行最佳操作。EpiCypher内部测试了修改后的CUT&Tag实验方案，结果发现新手和有经验的使用者的实验成功率都有所提高，这说明了CUT&Tag改进后的实用性。

 

✍ 这些实验操作流程的变化适用于正在使用第1版CUTANA™ CUT&Tag Kit或DIY CUT&Tag的科研人员吗?

是的。所有实验流程的更改都与CUTANATM CUT&Tag Kit的第1版以及 DIY CUT&Tag流程（https://www.epicypher.com/resources/protocols/cutana-pag-tn5-resources/）兼容。

您可以使用现有的试剂盒组分来按照第2版的实验流程操作，而不需要任何新材料！

 

需要注意的主要区别是试剂盒中去掉了TAPS Wash Buffer。之前使用TAPS Wash Buffer进行标记后洗涤，现在使用等体积的Pre-Wash Buffer洗涤。EpiCypher在该试剂盒中为使用者提供了充足的Pre-Wash Buffer来完成这一步骤。

 

【 总 结 】

CUTANA™ CUT&Tag Kit版本的更新反映了EpiCypher严格的研发工作。EpiCypher团队将继续完善CUT&Tag和CUT&RUN的相关研究，包括针对新应用和研究领域的优化。如果您有其他疑问，第2版的实验操作流程（https://www.epicypher.com/resources/protocols/cutana-cut-and-tag-kit-manual/）也许能解决您的问题，也欢迎联系EpiCypher中国代理商上海金畔生物咨询。

 

【 参考文献 】

1. Janssens DH et al. Scalable single-cell profiling of chromatin modifications with sciCUT&Tag. Nat Protoc 19, 83-112 (2024). https://doi.org/10.1038/s41596-023-00905-9.


2. Bartosovic M et al. Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues. Nat Biotechnol 39, 825-35 (2021). https://doi.org/10.1038/s41587-021-00869-9.


3. Wu SJ et al. Single-cell CUT&Tag analysis of chromatin modifications in differentiation and tumor progression. Nat Biotechnol 39, 819-24 (2021). https://www.doi.org/10.1038/s41587-021-00865-z.


4. Deng Y et al. Spatial-CUT&Tag: Spatially resolved chromatin modification profiling at the cellular level. Science 375, 681-6 (2022). https://doi.org/10.1126/science.abg7216.


5. Zhang D et al. Spatial epigenome-transcriptome co-profiling of mammalian tissues. Nature 616, 113-22 (2023). https://doi.org/10.1038/s41586-023-05795-1.


6. Henikoff S et al. Direct measurement of RNA Polymerase II hypertranscription in cancer FFPE samples. bioRxiv 2024.02.28.582647 (2024). https://doi.org/10.1101/2024.02.28.582647.


7. Henikoff S et al. Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag. Nat Commun 14, 5930 (2023). https://doi.org/10.1038/s41467-023-41666-z.


8. Kaya-Okur HS et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930 (2019). https://doi.org/10.1038/s41467-019-09982-5.


9. Kaya-Okur HS et al. Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc 15, 3264-83 (2020). https://doi.org/10.1038/s41596-020-0373-x.

如需了解更多详细信息或相关产品，

请联系EpiCypher中国代理商–上海金畔生物 

请联系EpiCypher中国代理商-上海金畔生物 

Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

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Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

核酸酶靶向切割和释放 (CUT&RUN)技术是由Steven henikoff博士团队开发的一种染色质图谱分析方法，基于Ulrich Laemmli博士的染色质免疫切割技术 (ChIC)，融合蛋白A与微球菌核酸酶 (pA-MNase)，选择性原位切割与抗体结合的染色质。在CUT&RUN中，细胞或细胞核固定化在固相载体上，从溶液中分离出pAG-MNase裂解的DNA片段。该方法与二代测序(NGS)兼容，可提供高质量的组蛋白翻译后修饰(PTMs)和染色质相关蛋白(如转录因子；Figure 1)。

Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

ChIP-seq是组蛋白PTMs和染色质相关蛋白全基因组定位的主要方法。在这种方法中，染色质通过超声或酶消化破碎，然后免疫沉淀目标特异性片段。尽管进行优化，但ChIP-seq需要大量细胞(通常为105 – 106个细胞)而且需要深度测序input 染色质与免疫沉淀物质(通常为 >3000万 reads/次)来从背景中解析信号。

ChIC和CUT&RUN通过将基因组片段靶向释放到溶液中，彻底改变了染色质调控。通过这一创新，背景显著减少，允许使用少量细胞且每个反应仅需300 – 800万reads/次对组蛋白PTMs和染色质相关蛋白进行高分辨率基因组图谱的绘制(Figure 2)。简化的工作流程和节省的成本使ChIC/CUT&RUN适用于高通量研究表观遗传生物学。


Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

FIGURE 2 Representative genome browser tracks show CUTANA™ CUT&RUN results using 500,000 K562   cells.  Clear peaks with the expected distribution profile are observed using 3-8 million sequencing  reads per reaction for a variety of epigenetic targets, including histone PTMs (H3K4me3, H3K27me3,  H3K27ac), transcription factors (CTCF), epigenetic reader proteins (BRD4), writer enzymes (MLL1),   and chromatin remodelers (SMARCA4).  Rabbit IgG antibody shown as a negative control (top track).

CUTANA™ChIC/CUT&RUN试剂盒包含48个反应的材料，专为多通道移液而设计，以实现CUT&RUN的高通量优势。该试剂盒包括阳性(H3K4me3)和阴性(Rabbit IgG)对照抗体，以及SNAP-CUTANA™ K-MetStat Panel (16个DNA条形码设计核小体携带广泛研究的赖氨酸甲基化PTMs)的分装分量。K-MetStat Panel加入到对照反应中，直接监测实验成功与否并帮助排除故障。此外，在pAG-MNase切割后，将剪切的E. coli DNA添加到所有反应中，以控制文库构建并使NGS标准化。该试剂盒与细胞和细胞核兼容，包括冻存和交联样品(Figure 3)。

Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

FIGURE 3 Heatmaps show CUT&RUN signal (red) and background (blue) of H3K4me3-enriched regions flanking annotated transcription start sites (TSS, +/- 2 kb). Gene rows are aligned across conditions, showing that genome-wide enrichment is preserved across sample types.


尽管建议从500,000个细胞开始，但只需使用5,000个细胞即可生成可比较的数据(Figure 4)。对照组的加入以及与不同靶类型、样本和低细胞数的兼容性，使该试剂盒成为染色质图谱实验的首选解决方案。

Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

FIGURE 4 Representative genome browser tracks for H3K4me3 (low abundance target) and H3K27me3  (high abundance target) CUT&RUN experiments using decreasing amounts of K562 cells. At 5,000   cells, data quality is largely indistinguishable from standard conditions (500,000 cells).

 保存条件 

OPEN KIT IMMEDIATELY and store components at room temperature, 4℃, and -20℃ as indicated (see User Manual corresponding to Kit Version 3). Stable for 6 months upon date of receipt.

Room Temperature (RT)

4℃

-20℃

8-strip Tubes

ConA Beads

5% Digitonin

0.5 M EDTA

E. coli Spike-in DNA

1 M Spermidine

100 mM Calcium Chloride

Bead Activation Buffer

SNAP-CUTANA™ K-MetStat Panel

SPRIselect Reagent

Manufactured by

Beckman Coulter Inc.

Pre-Wash Buffer

H3K4me3 Positive Control Antibody

0.1×TE Buffer

Stop Buffer

Rabbit IgG Negative Control Antibody

pAG-MNase

 数据示例 

Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

Figure 1: CUT&RUN DNA fragment size distribution analysis

CUT&RUN was performed as described in Figure 5. Library DNA was analyzed by Agilent Tapestation®. This analysis confirmed that mononucleosomes were predominantly enriched in CUT&RUN (~300 bp peaks represent 150 bp nucleosomes + sequencing adapters).
Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

FIGURE 2 SNAP-CUTANA™ K-MetStat Spikein Controls

DNA-barcoded designer  nucleosomes (dNucs) representing 16 K-methyl PTMs: mono-, di-, and tri-methylation at H3K4, H3K9, H3K27, H3K36, and H4K20, as well as  unmodified control, were spiked into CUT&RUN  reactions prior to the addition of antibodies (IgG, H3K4me3). Spike-in barcodes were counted and  normalized from raw fastq files using the shell  script and analysis sheet available at  epicypher.com/19-1002. Barcodes for IgG (top;  normalized to total reads) and H3K4me3 (bottom; normalized to on-target) antibodies are  shown. The spike-ins confirmed optimal  experimental conditions (H3K4me3 antibody  specifically recovered the target dNuc, while IgG  showed no preferential enrichment).
Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

Figure 3: CUT&RUN genome-wide heatmaps

CUT&RUN was performed as described in Figure 5. Heatmaps show two replicates (“Rep”) of IgG (left) and H3K4me3 (right) kit control antibodies in aligned rows ranked by intensity (top to bottom) and colored such that red indicates high localized enrichment and blue denotes background signal.
Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

Figure 4: Representative gene browser tracks

CUT&RUN was performed as described in Figure 5. A representative 174 kb window at the TRMT2A gene is shown for two replicates (“Rep”) of IgG and H3K4me3 kit control antibodies. Representative tracks are also shown for antibodies to H3K27me3 and the transcription factor CTCF. The CUT&RUN kit produced the expected genomic distribution for each target. Images were generated using the Integrative Genomics Viewer (IGV, Broad Institute).
Epicypher热销产品——CUTANA™ ChIC/CUT&RUN Kit

Figure 5: CUT&RUN methods

CUT&RUN was performed using the CUTANA™ ChIC/CUT&RUN Kit starting with 500k K562 cells with 0.5 µg of either IgG (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0041), H3K27me3 (EpiCypher 13-0055), or 0.125 µg of CTCF (EpiCypher 13-2014) antibodies in duplicate. Library preparation was performed with 5 ng of DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ Library Prep Kit (EpiCypher 14-1001/14-1002). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2×50 bp). Sample sequencing depth was 3.5 million reads (IgG Rep 1), 3.8 million reads (IgG Rep 2), 4.7 million reads (H3K4me3 Rep 1), 6.9 million reads (H3K4me3 Rep 2), 6.6 million reads (H3K27me3 Rep 1), 4.7 million reads (H3K27me3 Rep 2), 3.9 million reads (CTCF Rep 1) and 4.6 million reads (CTCF Rep 2). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and blacklist regions. 

 订购详情 

货号

产品名称

规格

14-1048

CUTANA™ ChIC/CUT&RUN Kit

48 Reactions

 

如需了解更多详细信息或相关产品，请联系EpiCypher中国代理商-上海金畔生物 

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

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EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

EpiCypher新推出的DYKDDDDK抗体，适用于利用FLAG®*标记的靶蛋白进行的研究。该抗体符合EpiCypher的“CUTANA Compatible”标准，可用于研究蛋白质-DNA互作的新技术，CUT & RUN和CUT & Tag。每个批次的CUTANA Compatible抗体均使用经EpiCypher优化的方案（https://www.epicypher.com/resources/protocols/），按照指定的方法进行测试，并确定产生的峰值所显示的基因组分布模式与已报道的靶标功能一致。在过表达 3xFLAG-GATA3的乳腺癌细胞中，该DYKDDDDK Tag 抗体产生与 GATA3 抗体高度重叠的 CUT&RUN 峰（图 2-3）[1]**。


*FLAG® is a registered trademark of Merck KGaA, Darmstadt, Germany.

**Thanks to Dr. Takaku (UND) for 3xFLAG-GATA3-3xHA MDA-MB-231 cells.

产品详情

产品名称：DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

宿主来源：Rabbit

实验应用：CUT&RUN, ELISA, WB, IP, ICC

免疫原：A synthetic peptide sequence (DYKDDDDK)

克隆性：Recombinant Monoclonal 

保存条件：Stable for 1 year at 4°C from date of receipt

验证数据

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 1: Defined nucleosome spike-ins provide an in-assay control for DYKDDDDK Tag antibody in CUT&RUN

CUT&RUN was preformed as described in Figure 7. CUT&RUN sequencing reads were aligned to the unique DNA barcodes corresponding to each nucleosome in the SNAP-CUTANA™ DYKDDDDK Tag Panel (EpiCypher 19-5001). Data are expressed as a percent relative to on-target recovery (DYKDDDDK Tag set to 100%) or total counts (IgG/GATA3). IgG/GATA3 show no preferential binding to unmodified or DYKDDDDK spike-in nucleosomes. DYKDDDDK Tag antibody selectively enriches the DYKDDDDK Tag spike-in nucleosome, validating the antibody in CUT&RUN.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 2: DYKDDDDK-tagged protein peaks in CUT&RUN

CUT&RUN was preformed as described in Figure 7. Heatmaps show DYKDDDDK Tag antibody-enriched peaks called for FLAG-GATA3 overexpressing cells (OE) in aligned rows relative to all other experimental conditions. Rows are ranked by intensity (top to bottom) and colored such that red indicates high localized enrichment and blue denotes background signal. A high degree of overlap is observed for DYKDDDDK and GATA3 antibodies as expected in the OE cells, while empty vector control (Ctrl) shows absence of FLAG and lower GATA3 enrichment representing endogenous protein. IgG shows low background signal. H3K4me3, a canonical mark of promoters, does not appear in regions of high GATA3 enrichment.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 3: CUT&RUN representative browser tracks for DYKDDDDK-tagged protein

CUT&RUN was preformed as described in Figure 7. Gene browser shots were generated using the Integrative Genomics Viewer (IGV, Broad Institute). Two of the top called peaks in FLAG-GATA3 overexpressing (OE) cells are shown. The peaks show the same distribution patterns as observed in the genome-wide heatmaps (Figure 2). IgG shows low background, H3K4me3 is unchanged between empty vector control (Ctrl) and OE cells, GATA3 peaks are more robust in OE cells, and peaks overlap between GATA3 and DYKDDDDK antibodies in OE cells. These results demonstrate the robustness and specificity of DYKDDDDK Tag antibody in CUT&RUN experiments targeting FLAG-tagged proteins.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 4: Western blot data

Western analysis of FLAG-tagged protein in lysates from non-transfected human HEK293 cells, HEK293 transfected with Met-FLAG-IDO1, HEK293 transfected with IDO1-FLAG-HIS (Internal Tag), HEK293 transfected with IDO1-HIS-FLAG (C-Terminal Tag), and SUP-M2 cells. EpiCypher DYKDDDDK Tag antibody was used at 1:1,000. An HRP-conjugated goat anti-rabbit IgG antibody (Fortis A120-101P) was used as a secondary antibody. Chemiluminescence exposure time was 10 seconds. Lower panel shows rabbit anti-RPS3A antibody results (Fortis A305-003A).

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 5: Immunoprecipitation data

EpiCypher DYKDDDDK Tag antibody (20 µL/mg lysate) was used to immunoprecipitate lysates isolated from HEK293 cells transfected with either Met-FLAG-IDO1 (A) or IDO1-HIS-FLAG (C-terminal tag; B). For blotting immunoprecipitated DYKDDDDK Tag, EpiCypher DYKDDDDK Tag antibody was used at 1:1,000. Chemiluminescence exposure time was 10 seconds.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 6: Immunocytochemistry data

FFPE sections of HEK293 cells expressing either FLAG-tagged nuclear protein (left) or FLAG-tagged cytoplasmic protein (right) using EpiCypher DYKDDDDK Tag antibody. An HRP-conjugated goat anti-rabbit IgG antibody (Fortis A120-501P) was used as a secondary antibody. The substrate used was DAB.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 7: CUT&RUN methods

CUT&RUN was performed on 500k MDA-MB-231 native cells either stably overexpressing 3xFLAG-tagged GATA3 [1] or containing vector control. Either DYKDDDDK Tag (0.05 µg), H3K4me3 (0.5 µg; EpiCypher 13-0041), GATA3 (0.5 µg; CST 5852), or IgG (0.5 µg, EpiCypher 13-0042) antibodies were used with the CUTANA™ ChIC/CUT&RUN Kit v3 (EpiCypher 14-1048). 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 (2×50 bp). Sample sequencing depth (vector control cells/overexpressing cells) was 5.6/4.5 million reads (IgG), 7.8/6.7 million reads (H3K4me3), 5.8/4.7 million reads (GATA3), and 6.2/5.5 million reads (DYKDDDDK Tag). 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

13-2031

DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

100 µL


参考文献

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

 

如需了解更多详细信息或相关产品，请联系EpiCypher中国代理商–上海金畔生物 

请联系EpiCypher中国代理商-上海金畔生物 

CUTANA™ 分析——CUT&RUN和CUT&Tag试剂盒及相关产品

发表于由

CUTANA™ 分析——CUT&RUN和CUT&Tag试剂盒及相关产品

CUT&RUN和CUT&Tag分析正在影响并改变目前的表观基因组学分析，其具有操作简单、使用细胞样本少和信噪比高等特点。与传统的ChIP-seq分析方法相比，CUTANA™分析方法具有多种优势：

√ 使用更少的细胞

√ 简化工作流程，没有染色质裂解及免疫沉淀反应

√ 性价比更高

CUTANA™ 分析——CUT&RUN和CUT&Tag试剂盒及相关产品

*CUTANA™分析仅用ChIP-seq所需的一小部分细胞和测序深度即可生成高质量的表观基因组图谱。上图分别为CUT&Tag(蓝色)，CUT&RUN(橙色)和ChIP-seq(灰色)在300 kb区域的实验结果。

相关产品

名称

货号

CUTANA™ ChIC/CUT&RUN Kit

14-1048

CUTANA™ CUT&Tag Kit

14-1102/14-1103

CUTANA™ CUT&RUN Library Prep Kit with Primer Set 1/2

14-1001/14-1002

更多产品详情，请联系EpiCypher全国代理-上海金畔生物 

CUTANA™ CUT&RUN Assays ——实现超敏基因组定位

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CUTANA™ CUT&RUN Assays ——实现超敏基因组定位

蛋白质和核酸是构成生命体最为重要的两类生物大分子，二者间的相互作用一直是分子生物学研究的中心问题之一。研究细胞内蛋白质-DNA相互作用的常用方法是染色质免疫沉淀(Chromatin Immunoprecipitation, ChIP) ，同时ChIP还常被用于确定基因组上与组蛋白修饰相关的特定位点（即组蛋白修饰酶的靶标）。但是由于ChIP存在高细胞需求量、技术难度大、成本高、深度测序、数据质量差以及变量大等缺点，在实际应用的过程中局限颇多。

核酸酶靶向切割和释放（Cleavage Under Targets and Release Using Nuclease, CUT&RUN）是表观遗传学的一种新型技术方法，在蛋白质-DNA的相互作用以及组蛋白翻译后修饰（PTMs）的基因组定位研究中取得突破性进步。CUT&RUN对传统的ChIP检测方法进行了重大的修改，消除ChIP固有的一些缺点，简化工作流程，跳过了ChIP-seq中包括染色质片段化和抗体pull down等非常具有挑战性步骤，用更少的细胞量和测序读数获取更佳的数据。

对于新用户，CUTANA™ CUT&RUN提供了您开展实验所需要的一切，包括简单易上手的实验套装、实验方案以及验证过的抗体等等，实验操作流程如下：

  CUT&RUN工作流程  

● 固定细胞
 CUTANA™ CUT&RUN Assays ——实现超敏基因组定位
● 添加抗体和pAG-MNase（Protein A-Protein G-微球菌核酸酶）
● 激活MNase并切割DNA
● 结合抗体的复合物扩散至溶液中
● 准备测序文库
● 测序

  为什么选择CUTANA™ CUT&RUN？

如下图所示，CUTANA™ CUT&RUN在使用更低测序读数的同时，结果要更优于ChIP-seq。

CUTANA™ CUT&RUN Assays ——实现超敏基因组定位

CUTANA™ CUT&RUN Assays ——实现超敏基因组定位

  CUTANA™ CUT&RUN优势：

● 节省了10倍的测序成本，省时高效。（与ChIP-seq相比）

● 低细胞需求量。（低至5k)

● 可作用于多种多样的靶标和样本类型，

● 操作流程简单易上手。

● 测序结果信噪比高。

● 实验可重复性好。


CUT&RUN可以用于研究多种类型的靶标，包括转录因子、染色质相互作用蛋白和组蛋白翻译后修饰，还为一些研究染色质重塑等十分具有挑战性的目标提供了机会。


下图为具有代表性的基因组浏览轨迹，展示了使用K562细胞的CUTANA CUT&RUN结果。通过将每个样本中约300-800万个测序reads用于表观遗传学的各靶标，可以观察到具有期望分布面的清晰峰。

CUTANA™ CUT&RUN Assays ——实现超敏基因组定位

  新手推荐产品：

CUTANATM CUT&RUN AND LIBRARY PREP KITS

EpiCypher的CUTANA™ CUT&RUN AND LIBRARY PREP KITS为用户提供了染色质定位实验的细胞-测序解决方案，该试剂盒包含了从细胞到测序流程中所有必要的对照以及验证试剂，充分确保实验能够获得高质量数据。

CUTANA™ CUT&RUN Assays ——实现超敏基因组定位

√ CUT&RUN首推产品！ 

√ 功能全面，新人必备！

√ 兼容性强：新鲜、冷冻或交联的细胞/细胞核均可使用！

CUTANATM REAGENTS

CUTANA™系列的所有试剂均经过测试和验证，适用于本公司CUTANA™ CUT&RUN优化后的工作流程。

CUTANA™ CUT&RUN Assays ——实现超敏基因组定位

√ 用于设计和执行制定的CUT&RUN实验

√ 产品齐全：ConA磁珠、pAG-MNase、大肠杆菌插入DNA、DNA纯

化试剂盒等均可单独购买。

CUTANATM CUT&RUN ANTIBODIES

CUTANA™ CUT&RUN Assays ——实现超敏基因组定位

√  均通过科学家的大量验证，性能稳定！

√  可用于各种染色质靶标，包括组蛋白翻译后修饰、转录

因子和染色质重塑。

√  定期查验新靶点！

  SNAP Spike-ins：表观基因组学中的核小体定量对照  

SNAP Spike-ins将含有DNA条形码且携带组蛋白（已翻译后修饰）的半合成核小体作为表观基因组学分析的定量峰值对照，提高了分析可靠性的同时，还实现了精确样本的标准化。此外，SNAP Spike-ins不仅优势多，而且使用范围广，可适用但不局限于以下方面：

√  与CUT&RUN、CUT&Tag、和ChIP-seq测定兼容；

√  抗体特异性分析验证，可原位操作；

√  监测分析性能，可持续监测检测情况；

√  定量样本比较，可直接定量读数；

√  故障排除实验。

CUTANA™ CUT&RUN Assays ——实现超敏基因组定位

 

上图为产品SNAP-CUTANA™ K-METSTAT PANEL，该产品包含了15个携带疾病相关赖氨酸甲基化修饰的dNucs（即含DNA条形码的半合成核小体）和一个未经修饰的dNucs对照。

 

如需了解更多详细信息或相关产品，请联系EpiCypher中国代理商-上海金畔生物 

Epicypher热销产品——CUTANA™ pAG-Tn5 for CUT&Tag

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Epicypher热销产品——CUTANA™ pAG-Tn5 for CUT&Tag

CUTANA pAG-Tn5是靶向剪切及转座酶(CUT&Tag)技术中进行高效绘制染色质特征的关键试剂。与ChIP-seq相比，CUT&Tag在降低细胞需求量和测序深度的信噪比方面进行了显著改进。CUTANA pAG-Tn5是一种高活性的E. coli转座酶突变体(Tn5)与蛋白A/G的融合产物，可与来自多种物种宿主的靶抗体兼容。它也没有表位标签，使其适合于标签介导的CUT&Tag(如FLAG、HA、TY1、V5等)。该产品经过高度纯化，已去除污染的E. coli DNA，并在细胞数量少的情况下进行复杂分析。为了每次实验的标准化以及抗体验证和反应监测，推荐使用SNAP-CUTANA™ nucleosome spike-ins(如EpiCypher 19-1002)。Tn5带有mosaic 接头，可在CUT&Tag中使用。


Adapters

Tn5ME-A: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’

Tn5ME-B: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’

Tn5ME-rev: 5’-[phos]CTGTCTCTTATACACATCT-3’


组分

50 mM HEPES-KOH pH 7.2, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% glycerol.


保存条件

Stable for one year at -20℃ from date of receipt. The protein is not subject to freeze/thaw under these conditions.

数据示例

Epicypher热销产品——CUTANA™ pAG-Tn5 for CUT&Tag

FIGURE 1: 

Protein gel data. CUTANA pAG-Tn5 for CUT&Tag (1 µg) was resolved via SDS-PAGE and stained  with Coomassie blue. The migration and molecular  weight of the protein standards are indicated. Uncharged  pAG-Tn5 monomer is 78.5 kDa, however once charged  with DNA, Tn5 dimerizes to a final complex weight of 191 kDa.

Epicypher热销产品——CUTANA™ pAG-Tn5 for CUT&Tag

FIGURE 2:

Size distribution of released chromatin. CUT&Tag was performed as described above. Recovered DNA was directly PCR amplified to produce sequenceready libraries. Agilent TapeStation traces for libraries derived from negative control IgG (top) and H3K27me3 (bottom) antibodies are shown. Excised DNA is highly enriched for mononucleosomes (peak at ~300 bp reflects ~150 bp insert size).

Epicypher热销产品——CUTANA™ pAG-Tn5 for CUT&Tag 

FIGURE 3: 

CUT&Tag data. CUT&Tag was performed as described above. Representative sequencing tracks obtained using CUTANA pAG-Tn5 show a 229 kb close up view of the LAMC3 gene. CUTANA pAG-Tn5 produced clear peaks with genomic distribution profiles consistent with the known biological functions of H3K4me3 and H3K27me3 as well as minimal background in the IgG negative control.

订购详情

货号

产品名称

规格

15-1017

CUTANA™ pAG-Tn5 for CUT&Tag

50 Reactions

15-1117

CUTANA™ pAG-Tn5 for CUT&Tag

250 Reactions

如需了解更多详细信息或相关产品，请联系EpiCypher中国代理商-上海金畔生物 

EpiCypher® SNAP-CUTANA™ Spike-in用户指南

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EpiCypher® SNAP-CUTANA™ Spike-in用户指南

EpiCypher® SNAP-CUTANA™ Spike-in User Guide

This User Guide describes EpiCypher’s quantitative nucleosome spike-in technology, or SNAP (Sample Normalization and Antibody Profiling) Spike-in Controls for CUTANA™ CUT&RUN and CUT&Tag assays.

Table of Contents

1. CUTANA™ CUT&RUN and CUT&Tag Assay Overview …………………………………………….2

2. SNAP-CUTANA™ Spike-in Controls: Overview and Advantages ……………………………..   4

3. Applications of SNAP-CUTANA™ Spike-ins for CUT&RUN and CUT&Tag ……………….    6

3.1. CUT&RUN/CUT&Tag assay optimization. ……………………………………………………………  6

3.2. Monitor experimental success. …………………………………………………………………………… 7

3.3. Identify high quality histone PTM antibodies. ………………………………………………………  10

3.4. Normalize data and quantitatively compare samples……………………………………………   12

4. Addition of SNAP-CUTANA™ Spike-ins to CUT&RUN and CUT&Tag reactions ……..      14

4.1. Adding the K-MetStat Panel to CUT&RUN reactions ……………………………………………  14

4.2. Adding the K-MetStat Panel to CUT&Tag reactions …………………………………………….   15

5. Analysis of SNAP-CUTANA™ Spike-in Control Sequencing Data ………………………….     16

5.1. Count the number of sequencing reads assigned to each nucleosome in the panel....  16

5.2. Generate a heatmap of the spike-in reads ………………………………………………………….  17

5.3. Examine spike-in data ……………………………………………………………………………………… 18

6. SNAP-CUTANA™ Spike-in Panel DNA Barcodes ………………………………………………….. 19

7. References …………………………………………………………………………………………………………20

1. CUTANA™ CUT&RUN and CUT&Tag Assay Overview

Cleavage Under Targets & Release Using Nuclease (CUT&RUN) and Cleavage Under Targets & Tagmentation (CUT&Tag) are revolutionary genomic mapping strategies developed by the group of Dr. Steven Henikoff1,2. Both assays build on the Chromatin ImmunoCleavage (ChIC) approach from Dr. Ulrich Laemmli3, in which Protein A/G is used to recruit an enzymatic domain to antibody-bound chromatin in situ3. An important feature of CUT&RUN/CUT&Tag is the immobilization of cells (or nuclei) to solid support (Figure 1), which streamlines assay workflows, improves signal-to-noise (vs. ChIP), and enables low cell inputs and sequencing requirements.

In CUT&RUN, a pAG-Micrococcal Nuclease (pAG-MNase) fusion is used to cleave antibodylabelled chromatin. Fragments are released from cells and purified (Figure 1A). In CUT&Tag, pAG is fused with prokaryotic transposase 5 (pAG-Tn5) to cleave and tagment antibody-bound chromatin with sequencing adapters (Figure 1B). Both assays are compatible with nextgeneration sequencing (NGS) to provide high quality profiles of histone post-translational modifications (PTMs) and chromatin-associated proteins. Robust CUT&RUN and CUT&Tag protocols, based on EpiCypher’s validated workflows, are available (epicypher.com/protocols).

EpiCypher® SNAP-CUTANA™ Spike-in用户指南

Figure 1: Overview of the CUTANA™ CUT&RUN (A) and CUT&Tag (B) protocols.

Improved controls for CUT&RUN and CUT&Tag experiments

Compared to Chromatin ImmunoPrecipitation sequencing (ChIP-seq), the leading chromatin mapping assay, CUT&RUN and CUT&Tag consistently generate higher quality data with improved signal-to-noise, while using a fraction of the cellular input and sequencing depth.

But how can users be sure these new assays work as intended? What controls are available?

Typical CUT&RUN/CUT&Tag experiments include positive (e.g. H3K4me3) and negative (e.g. IgG) control antibodies and validated cell lines (e.g. K562 cells). For both assays we also recommend confirming cell viability and bead binding, as well as the size distribution of final NGS libraries (described in the CUTANA™ CUT&RUN and CUT&Tag protocols at epicypher.com/protocols). However, even with such robust controls, questions remain:

• Is my histone PTM antibody (including H3K4me3 control antibody) specific? Our previous4 and ongoing work (chromatinantibodies.com) has shown that PTM antibodies frequently cross-react with related PTM targets and can exhibit application-specific performance. Through our extensive development of CUT&RUN/CUT&Tag assays to various targets, EpiCypher has found that antibodies that work well in ChIP may not always work in CUT&RUN and/or CUT&Tag. In-application testing against a defined panel of onand off-target nucleosome substrates is the ideal strategy to identify high qualityantibodies4.

• If my reaction fails or NGS data are of poor quality, how do I troubleshoot my experiment? CUT&RUN and CUT&Tag methods can be challenging, especially for new users, and it is not always clear which step requires optimization. For instance, poor signalto- noise in NGS data could be due to failed enzymatic activity, poorly optimized buffe conditions, antibody cross-reactivity, bead clumping, or problems with cell preparation, among others. Defined spike-in controls that replicate physiological chromatin structure (i.e. nucleosomes) can help optimize workflows and guide troubleshooting strategies.

EpiCypher is a leader in the development of semi-synthetic/recombinant nucleosomes and has recently leveraged this technology for the development of SNAP (Sample Normalization and Antibody Profiling) Spike-in Controls (epicypher.com/technologies/snap-spike-in-controls). Our recently launched SNAP-CUTANA™ K-MetStat Panel can be used as a quantitative spike-in control in both CUT&RUN and CUT&Tag reactions targeting histone lysine methylation PTMs.

In this User Guide we describe SNAP-CUTANA™ Spike-ins using the K-MetStat Panel (EpiCypher #19-1002) as our primary example. We review how to incorporate these spikeins into CUT&RUN/CUT&Tag approaches, data analysis, and interpretations. For additional product information, visit epicypher.com/products/nucleosomes/snap-cutana-spike-in-controls.

EpiCypher® SNAP-CUTANA™ Spike-in用户指南（完整版）下载链接：SNAP-CUTANA_K-MetStat_user_guide.pdf (epicypher.com)，

      扫描二维码下载：

EpiCypher® SNAP-CUTANA™ Spike-in用户指南

如需了解更多详细信息或相关产品，请联系EpiCypher中国代理商-上海金畔生物 

CUTANA CUT&RUN 2022重要文献精选

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CUTANA CUT&RUN 2022重要文献精选


CUT&RUN（Cleavage Under Targets and Release Using Nuclease）是一项用于在细胞天然染色质环境下检测蛋白质-DNA相互作用的强大技术。在CUT&RUN中，蛋白A和蛋白G与微球菌核酸酶(pAG-MNase)融合，并选择性地切割抗体标记的染色质，经过剪切后的片段从细胞中分离出来，纯化，最后通过NGS进行分析。作为ChIP的高效替代方案，CUT&RUN克服了传统ChIP-seq分析法的许多缺点。

 

与ChIP-seq相比CUT&RUN的优势 

● 需要的细胞数量较少：CUTANA™ CUT&RUN分析只需要5,000个细胞即可生成高分辨率的结果。

● 操作步骤简单：CUTANA™ CUT&RUN在3天内即可完成从细胞到文库的建立。还适用于多道移液器和8联排管，提高了分析的重复性和通量。

● 测序成本降低：只需要300 – 800万个测序读段，高通量测序可以检测更多样本。

● 减少实验中需要优化的步骤：CUT&RUN跳过了ChIP-seq中最具挑战性的部分(包括交联、染色质片段化和免疫沉淀(IP))，只需要较少的优化步骤。

 

EpiCypher可提供包括CUTANA CUT&RUN Assay Kit和 Library Prep Kit，以及一系列不断扩大的经过CUT&RUN验证的抗体产品。SNAP-CUTANA™ K-MetStat Panel提供了一个基本的检测控制，可用于抗体验证，实验流程的优化，并作为实验成功与否的直接衡量标准。下面重点介绍了一些研究，展示了CUTANA CUT&RUN在不同的研究领域的应用。希望这些文献能够为CUT&RUN如何应用于您的项目提供思路。

 

1. Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer–promoter interactions

 

Wang et al. Nucleic Acids Research, 2022. PMID: 35849129

细胞类型：Transfected HeLa cells and HEK293T cells

目标蛋白：H3K27ac

主要内容：argeted modification of the epigenome represents a promising strategy for precision medicine, since it allows activation of genes without disrupting DNA sequence. CRISPR/Cas9 has been cleverly modified for this purpose by fusing a nuclease-defective Cas9 (dCas9) with transcriptional activation domains, such as histone lysine acetyltransferases CBP or p300. Here, Wang et al. used CUT&RUN to help analyze local and genome-wide changes in chromatin structure when using different dCas9 activation systems. In agreement with previous studies, they found that dCas9 activators were highly variable, with results varying by the type of dCas9 fusion protein, cell type, and genomic target. They also discovered that targeting dCas9 activators to enhancers can induce reciprocal epigenomic changes at target promoters, leading to increased gene expression.

上榜理由：虽然dCas9激活系统提供了特定的基因激活，但表观基因组变化也是可能的。CUT&RUN做为一种快速可靠的染色质分析检测方法，可帮助分析使用不同dCas9激活系统时，染色质结构的局部和全基因组变化。

 

2. Histone H3 proline 16 hydroxylation regulates mammalian gene expression

Liu et al. Nature Genetics, 2022. PMID: 36347944

细胞类型：MDA-MB-231 cells (hypoxia-sensitive breast cancer cells) and 293T cells; includes experiments with and without knockdown of EGLN2 using the inducible CRISPR v2 system

目标蛋白：H3P16oh, EGLN2, KDM5A, H3K4me3

主要内容：Low oxygen (hypoxia) induces transcriptional changes that drive tumor growth and increase cancer severity. Although multiple studies show that chromatin is responsive to hypoxia and plays a role in these processes, additional information is needed to provide a cohesive mechanism. Here, Liu et al. defined a novel histone PTM directly linked to hypoxia: prolyl (proline) hydroxylation on histone H3, proline 16. As part of this work, the authors used CUTANA CUT&RUN to validate H3P16oh antibodies, characterize its enrichment, and determine its function during hypoxia. Their experiments revealed substantial crosstalk between H3P16oh and H3K4me3, establishing H3P16oh as a hypoxia-sensitive regulator of gene expression and cell proliferation in breast cancer cell lines.

上榜理由：这篇文章为如何使用CUTANA CUT&RUN分析法来研究新的PTMs提供了重要思路。Li等人还展示了组蛋白PTMs如何调节疾病中的染色质结构和基因表达，为表观遗传学靶向药物开发提供了支持。

 

3. Mapping cis-regulatory elements in human neurons links psychiatric disease heritability and activity-regulated transcriptional programs

Sanchez-Priego et al. Cell Reports, 2022. PMID: 35649373

细胞类型： Excitatory glutamatergic neurons and inhibitory GABAergic neurons derived from human pluripotent stem cells, with and without stimulation (membrane depolarization)

目标蛋白：H3K27ac (0, 30, 90 min), FOS (0, 2 hr)

主要内容：Genetic risk variants for psychiatric diseases are concentrated in cis-regulatory DNA, suggesting roles in cell-type specific gene expression. However, due to the inherent challenges of studying human brain tissues, these genomic regions remain largely unexplored. In this paper, Sanchez-Priego et al. generated large amounts of excitatory and inhibitory neurons using human pluripotent stem cells. They profiled cells using a variety of techniques, including CUT&RUN, ATAC-seq, and RNA-seq, to identify putative cis-regulatory elements (i.e. enhancers) associated with activity-dependent gene expression. To support the relevance of their results to human disease, the authors compared the list of candidate enhancers to a large database of psychiatric-disease risk variants, which revealed significant links to schizophrenia, ADHD, and bipolar disorder.

上榜理由：Sanchez-Priego等人没有只针对个体变异进行研究，而是采取了整体法，他们使用多种技术，包括CUT&RUN, ATAC-seq和RNA-seq对细胞进行分析，定义疾病相关细胞类型中的表观基因组元素，并与不同数据库进行比较引用。

 

4. Acute depletion of human core nucleoporin reveals direct roles in transcription control but dispensability for 3D genome organization

Zhu et al. Cell Reports, 2022. PMID: 36323253

细胞类型：HCT116 cell line (all targets), HeLa cell line (NUP93 only)

目标蛋白：NUP93, NUP35, NUP205, BRD4, SEC13

主要内容：The nuclear pore complex acts as the gateway to the nucleus in eukaryotic cells and is composed of ~30 different nucleoporin proteins (NUPs). There is ample evidence that the nuclear pore complex helps regulate 3D chromatin organization and transcription. However, the functions of NUP subunits in human cells are not defined. Here, Zhu et al. studied NUPs using multiple techniques, including CUTANA CUT&RUN, Hi-C, PRO-seq, and CRISPR/dCas9 tethering. CUT&RUN showed that NUP proteins were generally bound to active chromatin regions. NUP93 specifically associated with promoters and enhancers and directly regulated transcription (similar to Brown et al. and Ibarra et al.). Strikingly, the authors found that core NUP proteins were not required for 3D chromatin architecture, in contrast to leading hypotheses in the field.

上榜理由：CUTANA CUT&RUN的一个关键优势是能够在自然条件下定位大分子复合物的亚基，而不需要交联。值得注意的是，在哺乳动物细胞中研究NUP蛋白和核孔复合物一直具有挑战性，而利用CUT&RUN分析技术更能够快速的助力这项研究。

 

相关产品推荐

产品名称

货号

规格

CUTANA™ ChIC/CUT&RUN Kit

14-1048

48 Reactions

CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows

15-1016

50 Reactions

250 Reactions

CUTANA™ CUT&RUN Library Prep Kit

14-1001

48 Reactions

SNAP-CUTANA™ K-MetStat Panel

19-1002

50 Reactions

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

13-0041

100 μg

AR CUTANA™ CUT&RUN Antibody

13-2020

100 μL

JUN/c-Jun CUTANA™ CUT&RUN Antibody

13-2019

100 μL

EGFR CUTANA™ CUT&RUN Antibody

13-2018

100 μL

CHD4 CUTANA™ CUT&RUN Antibody

13-2016

100 μL

TP53/p53 CUTANA™ CUT&RUN Antibody

13-2015

100 μL

EZH2 CUTANA™ CUT&RUN Antibody

13-2026

100 μL

SP1 CUTANA™ CUT&RUN Antibody

13-2024

100 μL

ELF1 CUTANA™ CUT&RUN Antibody

13-2023

100 μL

Menin CUTANA™ CUT&RUN Antibody

13-2021

100 μL

Histone H4K20me3 Antibody, SNAP-Certified™ for CUT&RUN

13-0054

100 μg

Histone H3K27me1 Antibody, SNAP-Certified™ for CUT&RUN

13-0052

100 μg

HA Tag CUTANA CUTandRUN Antibody

13-2010

100 μg

CTCF CUTANA™ CUT&RUN Antibody

13-2014

100 μL

NCOA3/SRC3 CUTANA™ CUT&RUN Antibody

13-2013

100 μL

Estrogen Receptor Alpha (C-Terminal) CUTANA™ CUT&RUN Antibody

13-2012

100 μL

Estrogen Receptor Alpha (N-Terminal) CUTANA™ CUT&RUN Antibody

13-2011

100 μL

CHD3 CUTANA CUTandRUN Antibody

13-2009

100 μL

BRM/SMARCA2 CUTANA™ CUT&RUN Antibody

13-2006

100 μL

CHD1 CUTANA™ CUT&RUN Antibody

13-2008

100 μL

SNF2H/SMARCA5 CUTANA CUTandRUN Antibody

13-2007

100 μL

SNF2L/SMARCA1 CUTANA™ CUT&RUN Antibody

13-2005

100 μL

MLL1/KMT2A CUTANA™ CUT&RUN Antibody

13-2004

100 μL

BRD4 CUTANA CUTandRUN Antibody

13-2003

50μL

BRG1/SMARCA4 CUTANA™ CUT&RUN Antibody

13-2002

100 μL

FOXA1/HNF3A CUTANA™ CUT&RUN Antibody

13-2001

100 μL

CUTANA™ Rabbit IgG CUT&RUN Negative Control Antibody

13-0042

100 μg

Histone H3K4me2 Antibody, SNAP-Certified™ for CUT&RUN

13-0027

100 μg

Histone H3K9me1 Antibody, SNAP-Certified™ for CUT&RUN and ChIP

13-0029

100 μg

如需了解更多详细信息或相关产品，请联系EpiCypher中国代理商-上海金畔生物 

CUTANA™ CUT&Tag Kit添新品啦！助您轻松应对CUT&Tag实验

发表于由

CUTANA™ CUT&Tag Kit添新品啦！助您轻松应对CUT&Tag实验

CUTANA™ CUT&Tag Kit添新品啦！助您轻松应对CUT&Tag实验


EpiCypher推出新品CUTANA™ CUT&Tag Kit，为组蛋白翻译后修饰（PTM）的超灵敏定位提供了全面的解决方案。CUTANA™ CUT&Tag Kit使用专有的Direct-to-PCR策略，在一个管中即可完成从细胞到PCR文库扩增，无需传统的文库制备流程，最大限度地减少样本损失。与多道移液器兼容，提高了通量和重复性。


蛋白质翻译后修饰 (PTM) 通过功能基团或蛋白质的共价添加、调节亚基的蛋白水解切割或整个蛋白质的降解来增加蛋白质组的功能多样性。这些修饰包括磷酸化、糖基化、泛素化、亚硝基化、甲基化、乙酰化、脂质化和蛋白水解，几乎影响正常细胞生物学和发病机制的所有方面。染色质免疫共沉淀技术（ChIP）作为用于研究组蛋白的各种共价修饰与基因表达的常用技术，仍然具有相当的局限性。近年来，基因组学和表观遗传学领域引入了两种新颖的染色质分析方法，即核酸酶靶向切割和释放技术（CUT&RUN）与靶向剪切及转座酶技术（CUT&Tag），克服了ChIP法的诸多缺点，令研究人员能够用更低的成本、更高的效率得到信噪比更高、重复性更好的实验结果。


什么是CUT&Tag技术？

CUT&Tag全称Cleavage Under Targets and Tagmentation (CUT&Tag)，是基于CUT&RUN的新兴姊妹技术。在EpiCypher的CUT&Tag实验流程中，孵育结合好特异性抗体与靶标蛋白后，加入蛋白 A、蛋白G与Tn5的复合物（pAG-Tn5），使得转座体进入细胞并与抗体结合，间接地固定在靶蛋白上；激活Tn5酶的切割活性，将靶蛋白结合的DNA区域切断，从而达到提取DNA、进行PCR扩增、构建文库的目的。在Tn5上融合了protein A/G抗体结合功能域的转座体pAG-Tn5是该技术的核心，使研究者不必碎裂染色质并跳过了传统的文库准备步骤（end repair, adapter ligation）。

CUTANA™ CUT&Tag Kit添新品啦！助您轻松应对CUT&Tag实验

CUT&Tag技术有何特色？

精简化的工作流程跳过了传统ChIP-seq具有挑战性的步骤，包括染色质碎片和抗体下拉，用更少的细胞和测序读段生成高分辨率数据。

◆ 更短的实验周期

在两天内能够完成从细胞到文库的建立，而传统的ChIP-seq需要五天(或更长时间)。

CUTANA™ CUT&Tag Kit添新品啦！助您轻松应对CUT&Tag实验

◆ 更少的细胞需求量与更低的成本

CUTANA™CUT&Tag分析仅使用10000-100000个细胞核即可生成高分辨率的图谱，甚至可用于单细胞水平测序，便于单细胞或珍贵样本的分析；与传统ChIP-seq（需要约3000万次读取）相比，CUT&Tag检测仅需要500-800万次测序读段，节省实验成本。

CUTANA™ CUT&Tag Kit添新品啦！助您轻松应对CUT&Tag实验

◆ 更高的信噪比

CUTANA™CUT&Tag使用较少的起始细胞、更少的测序读段，也能得到较低的背景信号和较强的目的信号。

 

◆ 无需文库准备步骤

测序文库的准备工作既费时又昂贵。通过在抗体结合的目标位点添加测序适配器，能够实现跳过传统的文库准备步骤（end repair, adapter ligation），极大地简化了实验流程。EpiCypher的CUTANA™CUT&Tag分析通过直接从反应混合物中扩增标记DNA进一步简化了这一策略，实现在一个管中完成从细胞到PCR文库扩增。

 

产品信息

货号

品名

规格

14-1102     

CUTANA™ CUT&Tag Kit , Primer Set 1      

48 reactions      

14-1103    

CUTANA™ CUT&Tag Kit , Primer Set 2    

48 reactions    

 

CUTANA™ CUT&Tag试剂盒：用户友好的表观基因组分析工具

试剂盒内包括CUT&Tags实验所需所有组分与对照品：

✔ 对照抗体 (Control antibodies)

✔ spike-in对照 (Nucleosome spike-in controls)

✔ 详细的质控分析评价指标 (Detailed quality control metrics) 

✔ 故障排除小贴士 (Troubleshooting tips)

 

试剂盒组分（蓝色的为EpiCypher特有的组分）

名称

货号

pAG-Tn5

15-1017

H3K27me3 Positive Control Antibody

13-0055t

Rabbit IgG Negative Control Antibody

13-0042t

Anti-Rabbit Secondary Antibody

13-0047

SNAP-CUTANA™ K-MetStat Panel

19-1002t

ConA Beads

21-1401

Non-Hot Start 2X PCR Master Mix

15-1018

8-strip Tubes

10-0009t

4.5 M NaCl

21-1013

0.5 M EDTA

21-1014

1 M MgCl2

21-1015

TAPS Buffer

21-1016

SDS Release Buffer

21-1017

SDS Quench Buffer

21-1018

0.1X TE Buffer

21-1019

Pre-Wash Buffer

21-1020

Pre-Nuclear Extraction Buffer

21-1021

Bead Activation Buffer

21-1022

5% Digitonin

21-2023

1 M Spermidine

21-1024

SPRIselect reagent manufactured by 

Beckman Coulter, Inc

21-1404

Multiplexing Primers

14-1102 and 14-1103 each contain combinatorial dual 

indices for multiplexed sequencing of up to 48 reactions. 

Combine the kits to multiplex up to 96 reactions.

 

CUTANA™ CUT&Tag试剂盒特点

✔ 可对低数量的细胞进行可靠的分析

✔ 从细胞到测序仅需2天

✔ 与ChIP相比检测成本更低

✔ 独有的单管流程，最大限度提高回收率并简化您的工作流程


EpiCypher是一家成立于2012年的表观遗传学公司。从专有组蛋白肽阵列平台EpiGold™开始，EpiCypher开发了一系列同类产品。同时，EpiCypher是重组核小体制造和开发的全球领导者。利用其独有技术，不断添加高纯度修饰重组核小体（dNucs™）产品。dNuc™多样性的产品为破译组蛋白编码和加速药物开发提供了强大的工具。EpiCypher还将dNuc™技术广泛的应用于多种分析测定产品中，包括：SNAP-ChIP®Spike-in Controls（用于抗体分析和ChIP定量）， EpiDyne®底物（用于染色质重塑和抑制剂筛选及开发），dCyher™测定（用于探究表观遗传蛋白质-组蛋白PTM结合相互作用）。最近，EpiCypher还推出了针对ChIC、CUT&RUN和CUT&Tag的高灵敏度表观基因组图谱CUTANA™分析。


如需了解更多详细信息或相关产品，请联系EpiCypher中国代理商-上海金畔生物 

Epicypher热销产品——CUTANA™ CUT&RUN Library Prep Kit

发表于由

Epicypher热销产品——CUTANA™ CUT&RUN Library Prep Kit

核酸酶靶向切割和释放 (CUT&RUN)是建立在免疫探测技术上的染色质图谱分析方法。在CUT&RUN中，融合蛋白AG-微球菌核酸酶（pAG-MNase），选择性原位切割抗体结合的染色质（Figure 1）。该方法与二代测序（NGS）兼容，可提供组蛋白翻译后修饰（PTMs）和染色质相关蛋白（如转录因子[TFs]）的高分辨率全基因组图谱。

FIGURE 1 Overview of the CUTANA™ CUT&RUN protocol.


产品优势

 CUTANATM Library Prep Kit 是第一个专门为CUT&RUN分析开发的文库构建试剂盒。

•对CUT&RUN的方法进行了优化，对比多用途或ChIP-seq文库构建试剂盒更具优势。

•对于CUT&RUN产生的有限输入，工作流程非常稳定，为使用0.5-10 ng DNA的Illumina® NGS提供了高质量的文库。

•试剂盒包含CUT&RUN文库制备所需的所有材料（酶、引物、DNA纯化磁珠、缓冲液和PCR管）。

•可轻松搭配CUTANA™ CUT&RUN试剂盒（EpiCypher 14-1048）或CUT&RUN Protocol （EpiCypher.com/protocols）使用，实现工作流程集成，高通量检测和保证结果的可靠性，降低实验成本。

•可以有效制备组蛋白PTMs和染色质相关蛋白（如TFs）CUT&RUN DNA的文库。

 

Multiplexing Primers

Primer Set 1 includes i5 primers 1-8 and i7 primers 1-6 

Primer Set 1 includes i5 primers 1-8 and i7 primers 7-12

保存条件

OPEN KIT IMMEDIATELY and store components at room temperature and -20°C as indicated (see Kit Manual for full instructions). Stable for 6 months upon date of receipt.

Room Temperature (RT)

-20℃

8-strip Tubes

End Prep Enzyme

Ligation Enhancer

SPRIselectreagent manufactured by

Beckman Coulter, Inc.

End Prep Buffer

High Fidelity 2X PCR

Master Mix

0.1X TE Buffer

Adapter for Illumina®

U-Excision Enzyme

Ligation Mix

i5 and i7 Primers


数据示例

Epicypher热销产品——CUTANA™ CUT&RUN Library Prep Kit

FIGURE 1：

CUT&RUN DNA Fragment Size Distribution Analysis. CUT&RUN was  performed as described above. Library DNA was  analyzed by Agilent TapeStation® , which  confirmed that mononucleosomes were  predominantly enriched in CUT&RUN (~300 bp  peaks represent 150 bp nucleosomes +  sequencing adapters). Peaks at ~380 bp  correspond to the SNAP-CUTANA™ K-MetStat Panel of spike-in controls(EpiCypher 19-1002).

Epicypher热销产品——CUTANA™ CUT&RUN Library Prep Kit

FIGURE 2：

Representative Gene Browser Tracks. CUT&RUN was performed as described above. A representative 674 kb window at the SEPTIN5 gene is shown for three replicates (”Rep”) of IgG and H3K4me3 antibodies, as well as individual tracks for H3K27me3 and the transcription factor CTCF, demonstrating the robustness and reproducibility of the workflow with a variety of targets. Sequencing libraries prepared with the CUTANA CUT&RUN Library Prep kit produced the expected genomic distribution for each target. Images were generated using the Integrative Genomics Viewer (IGV, Broad Institute)

订购详情

货号

产品名称

规格

14-1001

CUTANA™ CUT&RUN Library Prep Kit with Primer Set 1

48 Reactions

14-1002

CUTANA™ CUT&RUN Library Prep Kit with Primer Set 2

48 Reactions

 


如需了解更多详细信息或相关产品，请联系EpiCypher中国代理商-上海金畔生物