CUT&Tag（Cleavage Under Targets and Tagmentation）是一种建立在CUT&RUN和ChIC基础上的创新型表观基因组分析测定方法。CUT&Tag检测方法由Dr. Steven Henikoff的实验室研发，并于2019年首次报道。CUT&Tag技术的核心是在Tn5上融合了protein A/G抗体结合功能域的转座体pAG-Tn5。

与ChIP-seq相比CUT&Tag的优势

● 更快：可以在两天内完成从细胞到文库的建立，而ChIP-seq需要五天(或更长时间)。

● 更少的细胞：CUTANA™CUT&Tag分析仅使用1000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。ChIP-seq需要数百万个细胞来进行高质量测序，不利于单细胞或珍贵样本的分析。

● 提高信噪比：即使使用较少的起始细胞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：通过在抗体结合的目标位点添加测序适配器，CUT&Tag可以跳过传统的库准备步骤(end repair, adapter ligation)，极大地简化了实验步骤。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，允许用户在一个管中从细胞到PCR文库扩增。

● 低成本：与ChIP-seq(需要约3000万次读取)相比，CUT&Tag检测仅需要300-800万次测序读取，节省了70%的成本。

2022年许多利用CUTANA™ CUT&Tag技术进行分析研究的文献被发表，快速的推进了表观遗传学的研究。下面是其中一些文献的简要示例，因其对EpiCypher CUT&Tag产品和实验步骤的独特应用而脱颖而出。希望这些文献能为您的实验提供一些灵感！

1. Tumor microenvironmental signals reshape chromatin landscapes to limit the functional potential of exhausted T cells

Ford et al. Science Immunology, 2022. PMID: 35930654

细胞类型： FACS-sorted tumor-infiltrating CD8+ T cells (exhausted cell states), effector CD8+ T cells from draining lymph nodes (control); all cells from mouse B16F10 melanoma mouse model

目标蛋白： H3K4me3, H3K27me3

主要内容： T cell exhaustion is an important mechanism associated with cancer immunotherapy resistance. Combating T cell exhaustion requires studying the entire cellular progression from progenitor to terminally differentiated exhausted T cells, including changes to chromatin structure. In this paper, Ford et al. applied ultra-sensitive CUTANA CUT&Tag technology to map open and repressive chromatin during exhausted T cell development. The reduced cell requirements of CUT&Tag enabled high-resolution profiling of multiple histone PTMs from the FACS-sorted cell populations, which revealed distinct chromatin features linked with T cell exhaustion. The authors used these data to identify new epigenetic-based strategies for reactivation of exhausted T cells, pointing to new avenues for immunotherapy-resistant cancer research.

上榜理由：本文强调了CUTANA CUT&Tag技术在分析罕见或小细胞群(包括流式细胞分析仪分选的免疫细胞)方面的优势。

2. Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro

Zhang et al. Nature Biotechnology, 2022. PMID: 35332340

细胞类型： Frozen PBMCs from healthy human donors

目标蛋白： H3K4me1, H3K4me2, H3K4me3, H3K27ac, H3K27me3, H3K9me3, Phospho-Rpb1 CTD (Ser2/Ser5)

主要内容： In this paper, Zhang et al. used CUTANA pAG-Tn5 to establish single-cell CUT&Tag-pro (scCUT&Tag-pro), a platform that merges CUT&Tag with detection of cell surface proteins in individual cells. For proof-of-concept, scCUT&Tag-pro was used to profile a collection of histone PTMs in PBMCs. Each experiment mapped one histone PTM per cell with simultaneous detection of common immune cell surface proteins. Individual cells across data sets could be grouped by cell surface markers, and then further separated by chromatin profiling results. These data were combined with publicly available scRNA-seq data to create single-cell “metaomic” profiles.

上榜理由：Zhang等人为异质细胞群中染色质状态的综合化分析提供了新的工具。同时还强调了CUTANA表观基因组技术如何应用于特定用户的平台开发。

3. Nuclear RIPK1 promotes chromatin remodeling to mediate inflammatory response

Li et al. Cell Research, 2022. PMID: 35661830

Bonus: Also uses EpiDyne-FRET chromatin remodeling assays!

细胞类型： Mouse embryonic fibroblasts from Ripk1 mutant mice (untreated, TNFα stimulation, TNFα stimulation + RIP1K inhibitor); spinal cord tissue from patients with amyotrophic lateral sclerosis (ALS) and healthy patients

目标蛋白： p-S166-RIPK1 (active RIPK1), SMARCC2, BRG1/SMARCA4, H3K4me1, H3K27ac

主要内容： Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) modulates cell responses to TNFα cytokine signaling and is linked to increased expression of inflammatory genes in neurodegenerative disease. Although several RIPK1 inhibitors have progressed to clinical trials, the mechanism connecting RIPK1 to transcriptional changes is still unclear. In this paper, Li et al. find that RIPK1 regulates SWI/SNF BAF chromatin remodeling activity at inflammatory genes, increasing chromatin accessibility and promoting gene expression. CUTANA CUT&Tag assays provided key data from cell lines, mouse models, and ALS patient samples, confirming co-localization of RIPK1 and SWI/SNF enzymes at TNFα-induced genes.

上榜理由：了解外部信号(如细胞因子)如何与染色质调控联系在一起，对于识别新的药物靶点和更有效的治疗非常重要。整个研究过程CUTANA CUT&Tag起到了很重要的作用。

4. Epigenetic reader SP140 loss of function drives Crohn’s disease due to uncontrolled macrophage topoisomerases

Amatullah et al. Cell, 2022. PMID: 35952671

细胞类型： Primary human macrophages (control and with siRNA-mediated SP140 knockdown)

目标蛋白： TOP1, TOP2A

主要内容： Genome-wide association studies (GWAS) for immune diseases have identified multiple risk variants associated with chromatin. Here, Amatullah et al. investigate the function of SP140, an epigenetic reader protein with variants in Crohn’s disease, chronic lymphocytic leukemia, and multiple sclerosis. Using a series of mass spectrometry, cell culture, and CUTANA CUT&Tag experiments, the authors demonstrate that SP140 suppresses topoisomerase activity in healthy cells. GWAS variants of SP140 lead to changes in chromatin accessibility, activation of developmental genes, and defective antimicrobial responses. Treatment of inflammatory bowel disease (IBD) models with topoisomerase inhibitors improved phenotypes specifically in SP140 mutant mice, supporting further exploration for personalized medicine applications.

上榜理由：免疫疾病的全基因组关联研究(GWAS)已经确定了与染色质相关的多种风险变异。Amatullah等人研究了SP140的功能，通过质谱、细胞培养和CUTANA CUT&Tag分析，证明了SP140抑制健康细胞中的拓扑异构酶活性。文章还强调了突变位点为治疗提供了潜在信息。

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