CUT&Tag技术在代谢组织细胞的实验操作

doi:10.16288/j.yczz.22-253

遗传 ›› 2022, Vol. 44 ›› Issue (10): 958-966.doi: 10.16288/j.yczz.22-253

CUT&Tag技术在代谢组织细胞的实验操作

张子寅^1,²(), 周燕萍^1,², 孟卓贤^1,²()

1. 浙江大学医学院病理学与病理生理学系,杭州 310058
2. 浙江大学医学院浙江省疾病蛋白质组学重点实验室,杭州 310058

收稿日期:2022-08-01 修回日期:2022-09-20 出版日期:2022-10-20 发布日期:2022-10-03
通讯作者: 孟卓贤 E-mail:ziyinzhang@zju.edu.cn;zxmeng@zju.edu.cn
作者简介:张子寅,硕士研究生,专业方向：病理与病理生理学。E-mail: ziyinzhang@zju.edu.cn
基金资助:
国家自然科学基金重大研究计划(91857110);国家重点研究发展计划(2018YFA0800403);国家重点研究发展计划(2016YFC1305303);国家自然科学基金(81670740);国家优秀青年科学基金(81722012);浙江省自然科学基金(LZ21H070001);浙江大学基础医学创新研究院、中央高校基本科研专项基金、杭州市医学重点学科建设基金编号(OO20200055);王宽诚教育基金资助

The protocol of CUT&Tag for metabolic tissue cells

Ziyin Zhang^1,²(), Yanping Zhou^1,², Zhuo-Xian Meng^1,²()

1. Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou 310058, China
2. Key Laboratory of Disease Proteomics of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou 310058, China

Received:2022-08-01 Revised:2022-09-20 Online:2022-10-20 Published:2022-10-03
Contact: Meng Zhuo-Xian E-mail:ziyinzhang@zju.edu.cn;zxmeng@zju.edu.cn
Supported by:
the Training Program of the Major Research Plan of the National Natural Science Foundation of China(91857110);the National Key Research and Development Program of China(2018YFA0800403);the National Key Research and Development Program of China(2016YFC1305303);the National Natural Science Foundation of China(81670740);the National Natural Science Fund for Excellent Young Scholars of China(81722012);the Zhejiang Provincial Natural Science Foundation of China(LZ21H070001);the Innovative Institute of Basic Medical Sciences ofZhejiang University, and the Fundamental Research Funds for the Central Universities, the Construction Fund of Medical Key Disciplinesof Hangzhou(OO20200055);K.C. Wong Education Foundation

摘要/Abstract

摘要：

染色体靶向切割和标签化(clevage under target and tagment, CUT&Tag)技术利用Tn5转座酶与Protein A/G的融合蛋白,引导Tn5酶至与靶蛋白结合的抗体附近,对靶蛋白结合的附近染色质区域进行切割,随后通过标签化处理对片段化染色质进行文库制备,并利用高通量测序技术获取特定位点或蛋白质结合位置的染色质信息。CUT&Tag技术在蛋白质和DNA相互作用的研究领域起到了重大作用,不仅可以了解组蛋白修饰发生的位置,而且可以明确转录因子结合的区域。相较于传统的染色质免疫沉淀高通量测序(chromatin immunoprecipitation- sequencing, ChIP-seq)技术,CUT&Tag技术具有信噪比高、可重复性好、实验周期短、细胞投入量低等优点,在早期胚胎发育、干细胞、肿瘤以及表观遗传学等研究领域体现出巨大优势。本文将针对CUT&Tag在代谢组织细胞(以小鼠原代胰岛细胞为例)的具体操作步骤进行描述,以提供一种研究代谢细胞的表观遗传学方法。

关键词: CUT&Tag, 组蛋白修饰, 操作步骤, 代谢组织细胞

Abstract:

Cleavage under target and tagment (CUT&Tag) is a technology that utilizes the fusion protein of Tn5 transposase and protein A/G which can guide Tn5 enzyme to the antibody bound to target protein and cleave the chromatin regions adjacent to target protein. Chromatin libraries are then tagged and sequenced by the high-throughput sequencing to obtain chromatin information at specific sites or protein binding locations. CUT&Tag technology plays an important role in the research of DNA and protein interactions. It can be used to understand the modifications of histone and the bindings of transcription factors. Compared with the traditional chromatin immunoprecipitation-sequencing (ChIP-seq) technology, the CUT&Tag has the strengths of high signal-to-noise ratio, good repeatability, short experimental period, and low cell input. It shows great advantages in early embryonic development, stem cells, cancer, epigenetics and other research fields. In this article, we described the protocol of CUT&Tag for metabolic tissue cells (mouse primary islet cells), to provide an epigenetic method for studying metabolic cells.

Key words: CUT&Tag, histone modification, protocol, metabolic cell

张子寅, 周燕萍, 孟卓贤. CUT&Tag技术在代谢组织细胞的实验操作[J]. 遗传, 2022, 44(10): 958-966.

Ziyin Zhang, Yanping Zhou, Zhuo-Xian Meng. The protocol of CUT&Tag for metabolic tissue cells[J]. Hereditas(Beijing), 2022, 44(10): 958-966.

图/表 6

表1

图1

图2

图3

图4

图5

参考文献 8

[1]	Kidder BL, Hu GQ, Zhao KJ. ChIP-Seq: technical considerations for obtaining high-quality data. Nat Immunol, 2011, 12(10): 918-922. doi: 10.1038/ni.2117 pmid: 21934668
[2]	Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG, Ahmad K, Henikoff S. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun, 2019, 10(1): 1930. doi: 10.1038/s41467-019-09982-5 pmid: 31036827
[3]	Henikoff S, Henikoff JG, Kaya-Okur HS, Ahmad K. Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation. eLife, 2020, 9: e63274. doi: 10.7554/eLife.63274
[4]	Kaya-Okur HS, Janssens DH, Henikoff JG, Ahmad K, Henikoff S. Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc, 2020, 15(10): 3264-3283. doi: 10.1038/s41596-020-0373-x pmid: 32913232
[5]	Wei Y, Li K, Lu DR, Zhu HX. Optimization of CUT&Tag product recovery and library construction method. Hereditas (Beijing), 2021, 43(4): 362-374.
	韦晔, 李科, 卢大儒, 朱化星. CUT&Tag产物回收和建库方法的优化. 遗传, 2021, 43(4): 362-374.
[6]	Taylor BL, Liu FF, Sander M. Nkx6.1 is essential for maintaining the functional state of pancreatic beta cells. Cell Rep, 2013, 4(6): 1262-1275. doi: 10.1016/j.celrep.2013.08.010 pmid: 24035389
[7]	Qiao JT, Zhang ZY, Ji SH, Liu TL, Zhang XN, Huang YM, Feng WL, Wang KL, Wang JY, Wang SS, Meng ZX, Liu M. A distinct role of STING in regulating glucose homeostasis through insulin sensitivity and insulin secretion. Proc Natl Acad Sci USA, 2022, 119(7): e2101848119.
[8]	Wang RR, Qiu XY, Pan R, Fu HX, Zhang ZY, Wang QT, Chen HD, Wu QQ, Pan XW, Zhou YP, Shan PF, Wang SS, Guo GJ, Zheng M, Zhu LY, Meng ZX. Dietary intervention preserves β cell function in mice through CTCF-mediated transcriptional reprogramming. J Exp Med, 2022, 219(7): e20211779.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

仪器与材料名称	品牌	货号
4-羟乙基哌嗪乙磺酸(HEPES)	Sigma-Aldrich	H4034
氯化钾(KCl)	Sigma-Aldrich	P5405
氯化钙(CaCl₂)	Sigma-Aldrich	C5670
氯化锰(MnCl₂)	Sigma-Aldrich	M1787
氯化钠(NaCl)	Sigma-Aldrich	S3014
氯化镁(MgCl₂)	Sigma-Aldrich	M2670
亚精胺(Spermidine)	Sigma-Aldrich	S0266
蛋白酶抑制剂(Protease inhibitor)	Sigma-Aldrich	5056489001
毛地黄皂苷(Digitonin)	Sigma-Aldrich	D141
二甲基亚砜(DMSO)	Sigma-Aldrich	D4540
牛血清白蛋白(BSA)	MPbio	LS.0219989980
十二烷基硫酸钠(SDS)	Yeasen	20106ES76
TrypLE Express Enzyme (1×)	Gibco	12604013
磷酸缓冲盐溶液(PBS)	Hyclone	SH30256.01B
蛋白酶K(Proteinase K)	Sigma-Aldrich	P2308
乙二胺四乙酸(EDTA)	沪试	10009717
TruePrep Amplify Enzyme	诺唯赞	TD601
DNA抽提试剂(PCI)	Solarbio	P1012
无水乙醇	沪试	10009218
三氯甲烷(氯仿)	沪试	10006818
Anti-acetyl-Histone H3 Antibody	Sigma-Aldrich	06-599
Normal Rabbit IgG	Cell Signaling Technology	2729
Anti-Rabbit IgG (H+L)	Sigma-Aldrich	SAB3700883
Hyperactive pG-Tn5 Transposase for CUT&Tag	诺唯赞	S602
Concanavalin A coated beads (ConA beads)	诺唯赞	N515-C1
VAHTS DNA Clean Beads	诺唯赞	N411
1.5 mL EP管	Axygen	MCT-150-C
0.2 mL PCR管	Axygen	PCR-02D-L-C
四维旋转混合仪	其林贝尔	BE-1100

[1]	王雅楠, 徐涛, 王万鹏, 张庆祝, 解莉楠. 表观遗传修饰在作物重要性状形成中的作用[J]. 遗传, 2021, 43(9): 858-879.
[2]	王天一, 王应祥, 尤辰江. 植物PHD结构域蛋白的结构与功能特性[J]. 遗传, 2021, 43(4): 323-339.
[3]	张競文,续倩,李国亮. 癌症发生发展中的表观遗传学研究[J]. 遗传, 2019, 41(7): 567-581.
[4]	杨旭琼, 吴珍芳, 李紫聪. 哺乳动物体细胞核移植表观遗传重编程研究进展[J]. 遗传, 2019, 41(12): 1099-1109.
[5]	潘云枫, 王演怡, 陈静雯, 范怡梅. 线粒体代谢介导的表观遗传改变与衰老研究[J]. 遗传, 2019, 41(10): 893-904.
[6]	刘辰东, 杨露, 蒲红州, 杨琼, 黄文耀, 赵雪, 朱砺, 张顺华. 运动对骨骼肌基因表达的表观遗传调控作用[J]. 遗传, 2017, 39(10): 888-896.
[7]	张轲, 冯光德, 张宝云, 向伟, 陈龙, 杨芳, 储明星, 王凭青. 表观遗传标记在猪分子育种中的研究与应用前景[J]. 遗传, 2016, 38(7): 634-643.
[8]	孙凌云, 李星逾, 孙志为. 原发性肝癌的表观遗传学及其治疗[J]. 遗传, 2015, 37(6): 517-527.
[9]	郭雨辰, 雷秉坤, 邓小龙, 余垚, 吕红. sgf73⁺在裂殖酵母中的大规模遗传筛选[J]. 遗传, 2014, 36(7): 723-731.
[10]	孙源超, 秦训思, 陈宏, 沈伟. 细胞自噬发生的表观遗传调节[J]. 遗传, 2014, 36(5): 447-455.
[11]	施子晗, 李泽琴, 张根发. 植物组蛋白赖氨酸化修饰参与基因表达调控的机理[J]. 遗传, 2014, 36(3): 208-219.
[12]	沈圣, 屈彦纯, 张军. 下一代测序技术在表观遗传学研究中的重要应用及进展[J]. 遗传, 2014, 36(3): 256-275.
[13]	赵金璇, 王芳, 徐峥嵘, 范怡梅. 表观遗传调控pre-mRNA的选择性剪接[J]. 遗传, 2014, 36(3): 248-255.
[14]	许力凡张记田志强吴玉章. 表观遗传学与肿瘤干细胞[J]. 遗传, 2013, 35(9): 1049-1057.
[15]	王维，孟智启，石放雄. 组蛋白修饰及其生物学效应[J]. 遗传, 2012, 34(7): 810-818.

CUT&Tag技术在代谢组织细胞的实验操作

The protocol of CUT&Tag for metabolic tissue cells

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 8

相关文章 15

编辑推荐

Metrics