CUT&Tag（Cleavage Under Targets and Tagmentation）是一種建立在CUT&RUN和ChIC基礎(chǔ)上的創(chuàng)新型表觀基因組分析測定方法。CUT&Tag檢測方法由Dr. Steven Henikoff的實(shí)驗(yàn)室研發(fā)，并于2019年首次報(bào)道。CUT&Tag技術(shù)的核心是在Tn5上融合了protein A/G抗體結(jié)合功能域的轉(zhuǎn)座體pAG-Tn5。

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與ChIP-seq相比CUT&Tag的優(yōu)勢

●?更快：可以在兩天內(nèi)完成從細(xì)胞到文庫的建立，而ChIP-seq需要五天(或更長時(shí)間)。

●?更少的細(xì)胞：CUTANA?CUT&Tag分析僅使用1000個(gè)細(xì)胞核即可生成高分辨率的圖譜，甚至可用于單細(xì)胞水平測序。ChIP-seq需要數(shù)百萬個(gè)細(xì)胞來進(jìn)行高質(zhì)量測序，不利于單細(xì)胞或珍貴樣本的分析。

●?提高信噪比：即使使用較少的起始細(xì)胞也能得到較低的背景信號和較強(qiáng)的目的信號。

●?無需文庫準(zhǔn)備步驟：通過在抗體結(jié)合的目標(biāo)位點(diǎn)添加測序適配器，CUT&Tag可以跳過傳統(tǒng)的庫準(zhǔn)備步驟(end repair, adapter ligation)，極大地簡化了實(shí)驗(yàn)步驟。EpiCypher的CUTANA CUT&Tag分析通過直接從反應(yīng)混合物中擴(kuò)增標(biāo)記DNA進(jìn)一步簡化了這一策略，允許用戶在一個(gè)管中從細(xì)胞到PCR文庫擴(kuò)增。

● 低成本：與ChIP-seq(需要約3000萬次讀取)相比，CUT&Tag檢測僅需要300-800萬次測序讀取，節(jié)省了70%的成本。

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2022年許多利用CUTANA? CUT&Tag技術(shù)進(jìn)行分析研究的文獻(xiàn)被發(fā)表，快速的推進(jìn)了表觀遺傳學(xué)的研究。下面是其中一些文獻(xiàn)的簡要示例，因其對EpiCypher CUT&Tag產(chǎn)品和實(shí)驗(yàn)步驟的獨(dú)特應(yīng)用而脫穎而出。希望這些文獻(xiàn)能為您的實(shí)驗(yàn)提供一些靈感！

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

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

細(xì)胞類型：?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

目標(biāo)蛋白：?H3K4me3, H3K27me3

主要內(nèi)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：本文強(qiáng)調(diào)了CUTANA CUT&Tag技術(shù)在分析罕見或小細(xì)胞群(包括流式細(xì)胞分析儀分選的免疫細(xì)胞)方面的優(yōu)勢。

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

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

細(xì)胞類型：?Frozen PBMCs from healthy human donors

目標(biāo)蛋白：?H3K4me1, H3K4me2, H3K4me3, H3K27ac, H3K27me3, H3K9me3, Phospho-Rpb1 CTD (Ser2/Ser5)

主要內(nèi)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等人為異質(zhì)細(xì)胞群中染色質(zhì)狀態(tài)的綜合化分析提供了新的工具。同時(shí)還強(qiáng)調(diào)了CUTANA表觀基因組技術(shù)如何應(yīng)用于特定用戶的平臺開發(fā)。

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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!

細(xì)胞類型：?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

目標(biāo)蛋白：?p-S166-RIPK1 (active RIPK1), SMARCC2, BRG1/SMARCA4, H3K4me1, H3K27ac

主要內(nèi)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：了解外部信號(如細(xì)胞因子)如何與染色質(zhì)調(diào)控聯(lián)系在一起，對于識別新的藥物靶點(diǎn)和更有效的治療非常重要。整個(gè)研究過程CUTANA CUT&Tag起到了很重要的作用。

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

Amatullah et al. Cell, 2022.?PMID:?35952671

細(xì)胞類型：?Primary human macrophages (control and with siRNA-mediated SP140 knockdown)

目標(biāo)蛋白：?TOP1, TOP2A

主要內(nèi)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：免疫疾病的全基因組關(guān)聯(lián)研究(GWAS)已經(jīng)確定了與染色質(zhì)相關(guān)的多種風(fēng)險(xiǎn)變異。Amatullah等人研究了SP140的功能，通過質(zhì)譜、細(xì)胞培養(yǎng)和CUTANA CUT&Tag分析，證明了SP140抑制健康細(xì)胞中的拓?fù)洚悩?gòu)酶活性。文章還強(qiáng)調(diào)了突變位點(diǎn)為治療提供了潛在信息。

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