Optimization and Evaluation of Tn5 Transposase Fusion Protein in CUT&Tag

doi:10.11983/CBB22091

Abstract

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

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

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22091

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

Figures/Tables 8

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

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)

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

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

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.

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

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.

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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