Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Summary

Cleavage under targets and tagmentation (CUT&Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&Tag strategy for the profiling of histone modifications in plants.

Abstract

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Introduction

Transcription factor binding DNA sites and open chromatin associated with histone modification marks serve critical functional roles in regulating gene expression and are the major focuses of epigenetic research¹. Conventionally, chromatin immunoprecipitation assay (ChIP) coupled with deep sequencing (ChIP-seq) have been used for the genome-wide identification of specific chromatin histone modification or DNA targets with specific proteins, and is widely adopted in the field of epigenetics². Cleavage under targets and tagmentation (CUT&Tag) technology was originally developed by the Henikoff Lab to capture the protein-affiliated DNA fragments throughout the genome⁵. When compared to ChIP, CUT&Tagcan generate DNA libraries at high resolution and exceptionally low background using a small number of cells with a simplified procedure³. To date, methods for the analysis of chromatin regions with specific histone modifications using CUT&Tag have been established in animal cells^4,5. Specifically, single-cell CUT&Tag (scCUT&Tag) has also been successfully developed for human tissues and cells⁶. However, due to the complexity of the cell wall and secondary metabolites, CUT&Tag is still technically challenging for plant tissues.

Previously, we reported a CUT&Tag protocol using intact nuclei isolated from allotetraploid cotton leaves⁴. To demonstrate the efficiency of nuclei isolation and DNA capture using plant tissue, the profiling procedure is presented here. The major steps include intact nuclei isolation, in situ incubation with the antibody for chromatin modification, transposase incubation, adaptor integration, and DNA library preparation. The troubleshooting focuses on the CUT&Tag library preparation for the plant nuclei isolation and quality control.

In this study, we present a refined CUT&Tag strategy for plants. Not only do we provide a step-by-step protocol for H3K4me3 profiling in cotton leaves, but we also provide an optimized methodology for plant nuclei isolation, including the strategy for selecting the concentration of detergent for proper cell lysis and the strategy to filter the nuclei. Detailed steps with critical comments are also included in this updated protocol.

Protocol

1. Prepare transposase and stock solutions (Day 1)

NOTE: In this part, the oligonucleotide adapters are complexed with Tn5 transposase to make active transposase.

1. Dilute primers (primer A, primer B, and primer C) to 100 µM concentration using the annealing buffer (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA) (refer to Table 1 for sequence information of primers and Table 2 for the recipes for working solutions). Store at -20 °C until use.
2. Set up the following two reactions in PCR tubes: Reaction 1 (adaptor AB), 10 µL of 100 µM primer A, 10 µL of 100 µM primer B; Reaction 2 (adaptor AC), 10 µL of 100 µM primer A, 10 of µL 100 µM primer C.
3. Anneal the adapters in the PCR machine using the following program: heat lid (102 °C), 75 °C for 15 min, 60 °C for 10 min, 50 °C for 10 min, 40 °C for 10 min, 25 °C for 30 min.
   NOTE: The adapters are partially double-stranded DNA molecules (concentration = 50 pmol/µL).
4. Set up the following reaction in a 1.5 mL centrifuge tube: 10 µL of pA-Tn5 transposase (500 ng/µL or 7.5 pmol/µL), 0.75 µL of adaptor AB (50 pmol/µL), 0.75 µL of adaptor AC (50 pmol/µL), 7 µL of coupling buffer. Pipette 20x gently to mix well and incubate at 30 °C in a water bath for 1 h. The final concentration of transposase = 4 pmol/µL. Store at -20 °C until use.
   ​NOTE: Molar ratio of adaptor AB: adaptor AC: transposase = 0.5:0.5:1.
5. Prepare the stock solutions according to the recipes provided in Table 2 and autoclave or filter the stock solutions to sterilize.
6. Autoclave and sterilize scissors, filter paper, a stainless-steel sieve, a mortar, pestles, etc.

2. Nuclei isolation (Day 2)

1. Pre-cool the centrifuge to 4 °C. For each 1 g of the starting material (cotton leaves), prepare 25 mL of nuclear isolation buffer A (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA), 20 mL of nuclear isolation buffer B (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA), 50 mL of nuclear wash buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail 0.1%), 1 mL of antibody buffer (50 mM Tris pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.5 mM spermidine, 1 mg/mL BSA, protease inhibitor cocktail 0.1%, 0.05% w/v digitonin). Let the tubes sit on ice until use.
2. Grind fresh leaves (~1 g) in liquid nitrogen to a fine powder and transfer to a 50 mL tube containing 25 mL of chilled nuclear isolation buffer A.
3. Mix the buffer solution gently and incubate the tube on ice for 5 min with gentle shaking to disperse the material evenly.
4. Centrifuge for 5 min at 500 rcf at 4 °C to form the cell pellet.
5. Decant the supernatant and resuspend the pellet with 20 mL of chilled nuclear isolation buffer B supplemented with 0.5% Triton X-100. Invert the tube gently to resuspend the pellet completely.
   NOTE: The 20 mL nuclear isolation buffer B is applicable for 1 g starting material, and this volume can be scaled accordingly.
   1. Perform a pilot assay to test the effect of series concentration of Triton X-100 (e.g., 2 mL nuclear isolation buffer B, each with 0.1%, 0.25%, 0.5%, and 1% Triton X-100 for 100 mg of grinded tissues). The appropriate concentration of Triton X-100 is indicated by an accumulation of white/gray nuclei at the bottom and a dark green supernatant after lysis and centrifuging at low speed (< 500 rcf for 4 min).
6. Filter the resulting cell lysate with a combination of two sieves: a 500-mesh sieve on the top to remove the larger tissue debris and a 1000-mesh sieve on the bottom to collect the nuclei.
   NOTE: Choose an appropriate mesh size for the sieves depending on the nuclei size of the plants.
7. Use sterile filter paper on the back side of the sieve to absorb small cell debris in the lysate.
8. Transfer the remaining nuclei containing lysate on the top side of the 1000-mesh sieve to four fresh 1.5 mL centrifuge tubes.
9. Centrifuge for 4 min at 300 rcf at 4 °C to collect the nuclei.
10. Remove as much of the supernatant as possible using a pipette tip and wash the pellet with nuclear wash buffer.
11. Add 800 µL of the nuclear wash buffer and invert the tubes gently to resuspend the pellet. Spin the tube again for 4 min at 300 rcf at 4 °C.
12. Perform a total of 3 washes.
13. (Optional) Image the nuclei under a microscope for DAPI staining.
    1. Transfer 100 µL of nuclei suspension in the nuclear wash buffer from Step 2.11. to a new 1.5 mL tube. Add 900 µL of nuclear wash buffer and 2 µL of DAPI stock solution (1 mg/mL) to make a final working concentration of 2 µg/mL for DAPI. Incubate the tube in the dark for 30 min at room temperature.
    2. After staining, centrifuge for 4 min at 300 rcf at 4 °C to collect the nuclei.
    3. Remove as much of the supernatant as possible using a pipette tip. Wash 3 times with 800 µL of the nuclear wash buffer.
    4. Remove as much of the supernatant as possible and resuspend the nuclei with 100 µL of nuclear wash buffer.
    5. Image the nuclei under a fluorescence microscope using the DAPI channel.
14. Combine the nuclei from two tubes (~50 µL of nuclei in volume in each 1.5 mL tube). Centrifuge for 4 min at 300 rcf at 4 °C. Remove as much of the remaining buffer as possible using a pipette tip.

3. Antibody incubation

1. Resuspend each 50 µL of nuclei in a 1.5 mL tube with 1 mL of antibody buffer.
2. Set up the following reactions in 1.5 mL tubes: two biological replicates of IgG control groups with 150 µL of nuclei (~ 1 µg of chromatin) and 2 µL of IgG (1 mg/mL) in each tube, two biological replicates of H3K4me3 assay groups with 150 µL of nuclei and 2 µL of anti-H3K4me3 antibody (1 mg/mL) in each tube.
3. Mix the solution gently and leave the tubes on a horizontal shaker for hybridization overnight at 4 °C and 12 rpm.
4. Pre-cool the centrifuge to 4 °C. Prepare fresh 50 mL of immunoprecipitation (IP) wash buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail 0.1%, ,0.05% w/v digitonin) in a 50 mL centrifuge tube. Let the tube sit on ice.
5. Centrifuge the tubes for 4 min at 300 rcf at 4 °C. Remove the antibody buffer from each tube.
6. Add 800 µL of IP wash buffer to each tube and mix gently. Leave the tubes on a horizontal shaker for 5 min at room temperature and 12 rpm.
7. After washing, centrifuge the tubes for 4 min at 300 rcf at 4 °C. Remove the supernatant using a pipette tip.
   NOTE: To avoid any disturbance of the nuclei pellet, leave ~50-80 µL of the buffer with the nuclei.
8. Repeat Steps 3.6.-3.7. two more times.
9. After the third wash, centrifuge for 2 min at 300 rcf at 4 °C. Remove the remaining buffer as much as possible.

4. Transposase incubation

1. Make fresh 1 mL of transposase incubation buffer (10 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail 0.1%, 0.05% w/v digitonin) by mixing 1 mL of the IP wash buffer with 30 µL of 5 M NaCl.
2. To prepare the Tn5 transposase mix for incubation, add 1 µL of the transposase to each 150 µL of the transposase incubation buffer.
   ​NOTE: Prepare the transposase solution mix first according to the number of reactions, and then add an aliquot of 150 µL to each tube.
3. Resuspend the nuclei with 150 µL of transposase solution.
4. Leave the tubes on a horizontal shaker at 12 rpm for 2-3 h at room temperature and mix every 10-15 min.
5. After transposase incubation, centrifuge the tubes for 4 min at 300 rcf at 4 °C. Remove the transposase incubation buffer in each tube.
6. Wash the tubes with IP wash buffer. To do so, add 800 µL of IP wash buffer to each tube, mix gently, and leave the tubes on a horizontal shaker at 12 rpm for 5 min at room temperature.
7. Centrifuge for 4 min at 300 rcf at 4 °C. Remove the supernatant using a pipette tip.
8. Repeat Steps 4.6.-4.7. two more times.
9. After the third wash, centrifuge for 2 min at 300 rcf at 4 °C. Remove the remaining buffer as much as possible.

5. Tagmentation

1. Freshly make 2 mL of the tagmentation buffer (10 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail 0.1%, 10 mM MgCl₂, 0.05% w/v digitonin) by mixing 2 mL of IP wash buffer with 60 µL of 5 M NaCl and 20 µL of 1 M MgCl₂.
2. Resuspend the nuclei with 300 µL of tagmentation buffer.
3. Mix the solution and incubate in a 37 °C water bath for 1 h for tagmentation.
4. Stop the tagmentation with 10 µL of 0.5 M EDTA (final concentration ~15 mM) and 30 µL of 10% SDS (final concentration 1%).

6. DNA extraction and NGS library construction

1. Add 300 µL of CTAB DNA extraction buffer as described⁷. Incubate the tubes in a 65 °C water bath for 30 min. Invert to mix every ~10 min.
2. Add 600 µL of phenol: chloroform: isoamyl alcohol (25:24:1) to each tube. Mix thoroughly.
3. Pre-centrifuge the phase lock gel tubes for 2 min at 13,000 rcf at 4 °C. Transfer the above solution to phase lock gel tubes.
4. Centrifuge for 10 min at 13,000 rcf at 4 °C.
5. Transfer the supernatant (~600 µL) to a new 1.5 mL tube.
6. Add 600 µL of chloroform to each tube. Mix thoroughly.
7. Centrifuge for 10 min at 13,000 rcf at 4 °C.
8. Transfer the supernatant (~600 µL) to a new 1.5 ml tube.
9. Add 12 µL of 100% ethanol and 2 µL of GlycoBlue Coprecipitant. Mix thoroughly and store at -20 °C for 1 h.
10. Centrifuge for 10 min at 13,000 rcf at 4 °C.
11. Decant the supernatant. Wash the DNA pellet using 75% (v/v) ethanol and centrifuge for 5 min at 13,000 rcf at 4 °C.
12. Decant the supernatant. Air-dry the DNA pellet and resuspend the DNA in 25 µL of ddH₂O.
13. Set up the PCR reaction as follows. For a total of 50 µL of reaction, add 24 µL of DNA, 11 µL of ddH₂O, 10 µL of 5x TAE buffer, 2 µL of P5 primer (10 µM), 2 µL of P7 primer (10 µM), and 1 µL of TruePrep Amplify Enzyme. PCR program: 72 °C for 3 min, 98 °C for 30 s; then 14 cycles of 98 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, followed by 72 °C for 5 min.
    NOTE: The first 72 °C for 3 min for extension cannot be skipped. Overamplification of the library will lead to a high level of PCR duplicates in the NGS. Start from 12-14 PCR cycles in PCR reaction for H3K4me3 when using ~1 µg of chromatin in each reaction.
14. Load 2 µL of the PCR products on 1.5% agarose gel for electrophoresis.
15. Purify the PCR products using commercial DNA purification magnetic beads.
16. Quantify the library with a fluorometer and agarose gel electrophoresis.
    NOTE: Effective concentration of the library should be >2 nM by qPCR to ensure good quality.
17. Perform next-generation sequencing (NGS) and data analysis.

Results

Figure 1 depicts the CUT&Tag workflow. Figure 2 shows the DAPI staining of the intact nuclei. The goal of the "nuclei isolation" step was to obtain the intact nuclei at a sufficient amount for the subsequent CUT&Tag reaction. Figure 3 shows the agarose gel electrophoresis of PCR products. The IgG negative control is required in parallel when setting up the experiment. Compared with the IgG control group, the bul...

Discussion

Here, we have described CUT&Tag, a technology for generating DNA libraries at high resolution and exceptionally low background using a small number of cells with a simplified procedure compared to chromatin immunoprecipitation (ChIP). Our success with H3K4me3 profiling in cotton leaves suggests that CUT&Tag, which was first designed for animal cells, can also be used for plant cells. Both the Tris buffer system commonly used for ChIP assay⁸ and the HEPES buffer system used for animal CUT&#...

Materials

Name	Company	Catalog Number	Comments
Antibody
Anti-H3K4me3	Millipore	07-473
Normal rabbit IgG	Millipore	12-370
Chemicals
Bovine Serum Albumin (BSA)			Make 10 mg/ml BSA stock solution. Store at -20°C
digitonin (~50% (TLC)	Sigma-Aldrich	D141	Make 5% digitonin stock solution (200 mg digitonin [~50% purity] to 2 mL DMSO). Note: Sterilize using a 0.22- micron filter. Store at -20°C
dimethyl sulfoxide (DMSO)
chloroform
ethylenediaminetetraacetic acid (EDTA)			Make 0.5 M EDTA (pH = 8.5) stock solution. Note: Making 100 mL of 0.5-M EDTA (pH = 8.5) requires approximately 2 g of sodium hydroxide (NaOH) pellets to adjust the pH
ethanol
GlycoBlue Coprecipitant (15 mg/mL)	Invitrogen	AM9516
magnesium chloride (MgCl2)			Make 1 M MgCl2 stock solution
protease inhibitor cocktail	Calbiochem	539133-1SET
potassium chloride (KCl)			Make 1 M KCl stock solution
phenol:chloroform:isoamyl alcohol (25:24:1,v:v:v)
sodium chloride (NaCl)			Make 5 M NaCl stock solution
spermidine			Make 2 M spermidine stock solution, store at -20°C.
sodium dodecyl sulfate (SDS)			Make 10% SDS stock solution. Note: Do not autoclave; sterilize using a 0.22-micron filter
Tris base			Make 1 M Tris (pH = 8.0) stock solution
Triton X-100			Make 20% Triton X-100 stock solution
Enzyme
Hyperactive pG-Tn5/pA-Tn5 transposase for CUT&Tag	Vazyme	S602/S603	Check the antibody affinity of the protein A or protein G that is fused with the Tn5. Generally speaking, proteins A and G have broad antibody affinity. However, protein A has a relatively higher affinity to rabbit antibodies and protein G has a relatively higher affinity to mouse antibodies. Select the appropriate transposase products that match your antibody.
TruePrep Amplify Enzyme	Vazyme	TD601
Equipment
Centrifuge	Eppendorf	5424R
PCR machine	Applied Biosystems	ABI9700
Orbital shaker	MIULAB	HS-25
NanoDrop One  spectrophotometer	Thermo Scientific	ND-ONE-W