CUT&Tag技术在代谢组织细胞的实验操作

doi:10.16288/j.yczz.22-253

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

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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

Abstract

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

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

Figures/Tables 6

Table 1

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References 8

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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

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The protocol of CUT&Tag for metabolic tissue cells

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