Tn5转座酶融合蛋白在CUT&Tag实验中的优化及评价

doi:10.11983/CBB22091

摘要/Abstract

摘要： Tn5是一种细菌转座子。经改造的Tn5能够高效地切割DNA, 同时连接上特定的接头序列, 因而广泛应用于高通量二代测序文库构建中。CUT&Tag (Cleavage Under Target & Tagmentation)是一种改进的研究蛋白质与DNA互作的技术, 具有重复性好、信噪比高及操作简便等优点。该技术采用pA (Protein A)或pG (Protein G)与Tn5形成融合蛋白, 定位于特定抗体(用于识别目标蛋白), 利用Tn5的特性, 在目标位点附近打断DNA的同时引入测序接头, 随后提取DNA, 再进行PCR扩增即可获得测序文库。但不同类型的抗体与pA或pG的亲和力不同, 因此限制了部分抗体在CUT&Tag技术中的应用。为克服这一局限, 该文构建了pG与Tn5的融合蛋白表达载体, 通过原核表达及亲和纯化的方式获得pG-Tn5重组蛋白; 并以RNA聚合酶II (Pol II)特异性抗体Pol II Ser5P (小鼠IgG1型抗体和兔IgG型抗体)为例, 在模式植物拟南芥(Arabidopsis thaliana)中评估pA-Tn5与pG-Tn5在不同类型抗体的CUT&Tag测序文库构建中的效果。结果表明, IgG1型抗体与pG-Tn5的亲和力更高, 构建的文库质量更好, 而IgG型抗体与2种酶的亲和力相当; 同时, 较低起始量的植物材料也能获得较好的效果, 证明了CUT&Tag的应用优势。该研究优化了CUT&Tag技术, 可为后续CUT&Tag实验中针对不同抗体时Tn5融合蛋白的选择提供参考。

关键词: ChIP, CUT&Tag, RNA聚合酶II, Tn5

Abstract: Tn5 is a bacterial transposon. The engineered Tn5 can efficiently tag DNA while adding the adapter sequences. Therefore, it has been widely used in the preparation of high-throughput sequencing libraries. Cleavage Under Target & Tagmentation (CUT&Tag) is an improved technology for studying the interaction between protein and DNA, which has the advantages of good repeatability, high signal-to-noise ratio, and easy operation. This technology uses Protein A (pA) or Protein G (pG) and Tn5 to form a fusion protein, which can locate specific antibodies (the antibody is used to identify the target protein) and break the DNA near the target site while introducing sequencing adapters. Then, DNA was extracted, followed by PCR amplification to obtain the sequencing library. However, different types of antibodies have different affinities for pA and pG, thus limiting the application of CUT&Tag for some antibodies. To overcome this limitation, the expression vector of pG-Tn5 was constructed by recombination, and pG-Tn5 recombinant protein was obtained by prokaryotic expression and affinity purification. We used RNA polymerase II (Pol II)-specific antibodies (Pol II Ser5P, mouse IgG1 and rabbit IgG) to compare the efficiency of pA-Tn5 and pG-Tn5 in library preparation of CUT&Tag in Arabidopsis. The results showed that the IgG1 antibody had higher affinity for pG-Tn5, and the quality of the constructed library was better when pG-Tn5 was used. The rabbit IgG antibody has comparable affinities to the two enzymes. A lower starting amount of plant material can be applicable in CUT&Tag. This study provides a reference for the selection of Tn5 fusion proteins against different antibodies in future CUT&Tag experiments.

Key words: ChIP, CUT&Tag, RNA polymerase II, Tn5

刘晟宇, 刘晓斌, 朱家富, 苏京, 董志诚, 刘敏. Tn5转座酶融合蛋白在CUT&Tag实验中的优化及评价. 植物学报, 2023, 58(4): 602-611.

Shengyu Liu, Xiaobin Liu, Jiafu Zhu, Jing Su, Zhicheng Dong, Min Liu. Optimization and Evaluation of Tn5 Transposase Fusion Protein in CUT&Tag. Chinese Bulletin of Botany, 2023, 58(4): 602-611.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22091

https://www.chinbullbotany.com/CN/Y2023/V58/I4/602

图/表 8

表1 缓冲液

Table 1 Buffer used in this study

Buffer	Component	Concentration	Buffer	Component	Concentration
HEGX buffer	NaCl	0.8 mol·L^-1	HBC buffer	Tris-HCl, pH7.5	20 mmol·L^-1
	Glycerol	10%		Sucrose	352 mmol·L^-1
	TritonX-100	0.2%		MgCl₂	8 mmol·L^-1
	HEPES-KOH, pH7.2	20 mmol·L^-1		TritonX-100	0.08%
	EDTA	1 mmol·L^-1		β-mercaptoethanol	8 mmol·L^-1
Storage buffer	NaCl	0.8 mol·L^-1		Glycerol	20%
	Glycerol	10%	10× binding buffer	HEPES-KOH, pH8.0	20 mmol·L^-1
	Tris-HCl, pH7.5	50 mmol·L^-1		KCl	100 mmol·L^-1
	EDTA	0.2 mmol·L^-1		CaCl₂	10 mmol·L^-1
	DTT	2 mmol·L^-1		MnCl₂	10 mmol·L^-1
Annealing buffer	Tris-HCl, pH7.0	5 mmol·L^-1		Spermidine	5 mmol·L^-1
	EDTA	0.5 mmol·L^-1	Blocking buffer	HEPES-KOH, pH7.5	20 mmol·L^-1
	NaCl	50 mmol·L^-1		NaCl	150 mmol·L^-1
5× Tagmentation buffer	TAPS-NaOH, pH8.5	50 mmol·L^-1		Spermidine	0.5 mmol·L^-1
	MgCl₂	25 mmol·L^-1		BSA	0.1%
Lysis buffer	HEPES-KOH, pH7.2	50 mmol·L^-1		EDTA	2 mmol·L^-1
	NaCl	150 mmol·L^-1	Wash buffer	HEPES-KOH, pH7.5	20 mmol·L^-1
	EDTA	1 mmol·L^-1		NaCl	150 mmol·L^-1
	TritonX-100	1%		Spermidine	0.5 mmol·L^-1
	Glycerol	10%		BSA	0.1%
	β-mercaptoethanol	5 mmol·L^-1		Pepstatin A	1 μg·mL^-1
	Pepstatin A	1 μg·mL^-1		Aprotinin	1 μg·mL^-1
	Aprotinin	1 μg·mL^-1		PMSF	1 mmol·L^-1
	PMSF	1 mmol·L^-1	TE buffer	Tris-HCl, pH8.0	1 mmol·L^-1
HBB buffer	Tris-HCl, pH7.5	25 mmol·L^-1		EDTA	0.1 mmol·L^-1
	Sucrose	0.44 mol·L^-1	Dissolution buffer	NaCl	300 mmol·L^-1
	MgCl₂	10 mmol·L^-1		EDTA	1 mmol·L^-1
	TritonX-100	0.1%
	β-mercaptoethanol	10 mmol·L^-1

图1 pG/Tn5表达载体的构建及pA-Tn5和pG-Tn5的制备 (A) 利用PCR及同源重组的方式构建pG/Tn5; (B) pA-Tn5利用几丁质亲和标签的亲和纯化(泳道1-4为纯化前的蛋白混合物, 表示纯化前带有几丁质结合结构域和intein的pA-Tn5蛋白; 泳道5-8为洗脱后纯度较高的pA-Tn5, 表示纯化后的蛋白; 泳道9: 蛋白分子量标准); (C) pG-Tn5利用几丁质亲和标签的亲和纯化(泳道1-5为纯化前的蛋白混合物, 表示纯化前的蛋白; 泳道6-8为洗脱后纯度较高的pG-Tn5, 表示纯化后的蛋白; 泳道9: 蛋白分子量标准)

Figure 2 Construction of the pG/Tn5 expression vector and preparation of pA-Tn5 and pG-Tn5 (A) pG/Tn5 was constructed by PCR and homologous recombination; (B) pA-Tn5 was affinity purified by the chitin-binding domain (lanes 1-4 are the protein mixture before purification, represents the protein before purification; lanes 5-8 are pA-Tn5 with high purity after elution, represents the purified protein; lane 9: Protein molecular weight marker); (C) pG-Tn5 is affinity purified by chitin-binding domain (lanes 1-5 are the protein mixture before purification, represents the protein before purification; lanes 6-8 are pG-Tn5 with high purity after elution, represents the purified protein; lane 9: Protein molecular weight marker)

图2 pA-Tn5和pG-Tn5的活力测定泳道1: 大豆基因组DNA+Tn5; 泳道3: 大豆基因组DNA+pA- Tn5; 泳道5: 大豆基因组DNA+pG-Tn5; 泳道2、4和6: 大豆基因组DNA; 泳道7: Marker

Figure 2 Evaluation of the activities of pA-Tn5 and pG-Tn5 Lane 1: Soybean genomic DNA+Tn5; Lane 3: Soybean genomic DNA+pA-Tn5; Lane 5: Soybean genomic DNA+pG- Tn5; Lanes 2, 4 and 6: Soybean genomic DNA without enzyme; Lane 7: Marker

表2 实验组设计

Table 2 Experimental group design

Group	Enzyme type	Antibody	Dosage of Tn5 (μg)	Dosage of plant material (μL)
1	pA-Tn5	Ser5P Mouse IgG1	0.2	150
2	pA-Tn5	Ser5P Mouse IgG1	1	150
3	pG-Tn5	Ser5P Mouse IgG1	0.2	150
4	pG-Tn5	Ser5P Mouse IgG1	1	150
5	pG-Tn5	IgG	0.2	150
6	pG-Tn5	IgG	1	150
7	pA-Tn5	Ser5P Rabbit IgG	0.5	30
8	pA-Tn5	Ser5P Rabbit IgG	1	150
9	pG-Tn5	Ser5P Rabbit IgG	0.5	30
10	pG-Tn5	Ser5P Rabbit IgG	1	150
11	pA-Tn5	IgG	1	150
12	pG-Tn5	IgG	1	150

图3 插入片段长度分布 (A) 实验组4插入片段长度分布; (B) 实验组6插入片段长度分布; (C) 实验组8插入片段长度分布; (D) 实验组10插入片段长度分布。Group 4、6、8和10同表2。

Figure 3 Library insert size (A) Library insert size in experimental group 4; (B) Library insert size in experimental group 6; (C) Library insert size in experimental group 8; (D) Library insert size in experimental group 10. Group 4, 6, 8 and 10 are the same as shown in Table 2.

图4 CUT&Tag信号在基因上的平均分布 (A) 小鼠IgG1型抗体的CUT&Tag信号在基因上的平均分布; (B) 兔IgG型抗体的CUT&Tag信号在基因上的平均分布。TSS: 转录起始位点; TES: 转录终止位点

Figure 4 Average distribution of CUT&Tag signals on genes (A) Average distribution of CUT&Tag signals on genes of mouse IgG1 antibody; (B) Average distribution of CUT&Tag signals on genes of rabbit IgG antibody. TSS: Transcription start site; TES: Transcription end site

图5 CUT&Tag信号在染色体上的分布 CDS: 蛋白质编码区; 5′UTR: 5′非翻译区; 3′UTR: 3′非翻译区。TSS和TES同图4。Group 2、3、4、6、7、8、9和10同表2。

Figure 5 Distribution of CUT&Tag signals on chromosome CDS: Coding sequence; 5′UTR: 5′ untranslated region; 3′UTR: 3′ untranslated region. TSS and TES are the same as shown in Figure 4. Group 2, 3, 4, 6, 7, 8, 9 and 10 are the same as shown in Table 2.

图6 利用IGV展示CUT&Tag信号在个别基因上的分布 (A) 基因AT4G14880-AT4G14900区段的CUT&Tag信号; (B) 磷响应蛋白基因EXO的CUT&Tag信号; (C) 蓝光受体基因CRY1的CUT&Tag信号。从上至下分别对应实验组2-12的实验数据。

Figure 6 Distribution of CUT&Tag signals on individual genes by IGV (A) CUT&Tag signal of gene AT4G14880 to AT4G14900; (B) CUT&Tag signals of phosphorus responsive protein gene EXO; (C) CUT&Tag signals of blue light receptor gene CRY1. From top to bottom, it corresponds to the experimental data of experimental groups 2-12.

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