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.
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.
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 cells4,5. 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.
1. Prepare transposase and stock solutions (Day 1)
NOTE: In this part, the oligonucleotide adapters are complexed with Tn5 transposase to make active transposase.
2. Nuclei isolation (Day 2)
3. Antibody incubation
4. Transposase incubation
5. Tagmentation
6. DNA extraction and NGS library construction
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 bulk of the DNA fragments pulled with H3K4me3 antibody sample ranged from ~280 to 500 base pairs. Figure 4 shows the resulting next-generation sequencing for the anti-H3K4me3 antibody compared to the IgG negative control groups.
Figure 1: The workflow of CUT&Tag. This figure ismodified from Tao et al.4. Please click here to view a larger version of this figure.
Figure 2: DAPI staining of intact nuclei isolated from cotton leaves. Scale bar = 20 µM. Please click here to view a larger version of this figure.
Figure 3: Agarose gel electrophoresis of 2 µL of PCR products. Please click here to view a larger version of this figure.
Figure 4: Representative IGV screenshot for H3K4me3 signals. (A) Representative IGV overview of CUT&Tag signals across a large genome region. ~1500 kb genome regions were randomly selected. (B) Representative IGV screenshot for genes with varied expression levels showed high resolution of CUT&Tag signals. The normalized bigWig files generated from bamCompare by comparing the treatment bam file (CUT&Tag anti-H3K4me3 reaction) and the control bam file (IgG) were used. TPM, transcripts per kilobase of exon model per million mapped reads. This figure is modified from Tao et al.4. Please click here to view a larger version of this figure.
Table 1: Primer sequences. Please click here to download this Table.
Table 2: Buffer and solution recipes. Please click here to download this Table.
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&Tag work for CUT&Tag using plant nuclei4,9. The isolation of intact plant nuclei is the critical step for the successful application of CUT&Tag in plants. In the previously reported methodology for nuclei isolation4, the solution of grinded tissues was filtered through two layers of Miracloth before cell lysis. We found the efficiency of obtaining adequate nuclei reduced when using relatively aged leaves (e.g., leaves from 2-month-old cotton plants) and cotton fibers (e.g., 20-DPA fiber) because Miracloth retains most of the grinded tissues. In this video protocol, the plant nuclei isolation was optimized by filtering the cell lysate through a 500-mesh (20 µM pore size) stainless steel sieve. This altered operation significantly improved the efficiency of removing larger tissue debris and obtaining adequate nuclei for the subsequent reaction.
After PCR for library construction (Step 6.13.), the DNA fragments can be purified and then profiled by NGS. However, if the IgG control group also depicts an enrichment in small fragments, it indicates a strong background of random DNA cutting by transposase, which may be caused by damaged nuclei or insufficient washing for the removal of unbound antibody or transposase. In this case, the parallel samples for specific antibodies were not recommended for further NGS.
Recently, single-cell CUT&Tag by adapting the droplet-based 10x Genomics single-cell ATAC-seq platform has been developed and applied in profiling H3K4me3, H3K27me3, H3K27ac, and H3K36me3 histone modifications. In a study on the chromatin occupancy of transcription factor OLIG2, the cohesin complex component RAD21 in the mouse brain provided unique insights into epigenomic landscapes in the central nervous system6. Thus, CUT&Tag can be applied to examine the epigenomic landscapes for both bulk histone modification and the specific TFs with low input requirements9, even at the single-cell level6. These applications of CUT&Tag indicate that the study of plant epigenetic regulation in coordinating gene functional networks during the dynamics of development and environmental responses can be precise in the temporal and spatial pattern.
CUT&Tag is a method still under developmental processes with problems that need to be addressed. For the transcription factors that are not abundantly expressed or are weakly, transiently, or indirectly bound to chromatin10, the differences between crosslinked and native nuclei in CUT&Tag need to be compared. The CUT&Tag strategy for profiling with transcription factors at low abundance is still technically challenging. In addition, due to the limited availability of protein epitope, most ChIP antibodies that are validated to work with crosslinking conditions may not work well with native conditions in CUT&Tag; the sensitivity and specificity of antibodies need to be validated. Furthermore, the Tn5 transposase used in CUT&Tag has high affinity to open-chromatin regions11, thus CUT&Tag might introduce bias, which also needs to be addressed in future studies.
The authors have nothing to disclose.
This work was financially supported in part by grants from the National Natural Science Foundation of China (NSFC, 31900395, 31971985, 31901430), and Fundamental Research Funds for the Central Universities, Hainan Yazhou Bay Seed Lab (JBGS, B21HJ0403), Hainan Provincial Natural Science Foundation of China (320LH002), and JCIC-MCP project.
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 |