Protocol to investigate bivalent histone modification dynamics via chromatin immunoprecipitation followed by re-chromatin immunoprecipitation

Summary

Epigenetic modifications play key roles in regulating transcription by altering chromatin structure and accessibility in response to internal cues or external stimuli. Here, we present a protocol for assessing bivalent histone modification changes through chromatin immunoprecipitation (ChIP)-reChIP assay in response to warm ambient temperature in Arabidopsis. We describe steps to perform transient warm ambient temperature treatment on Arabidopsis seedlings. We then detailed procedures for ChIP-reChIP, which can be applied to investigate various DNA-protein interaction dynamics in different experimental contexts.

For complete details on the use and execution of this protocol, please refer to Shao et al.¹

Graphical abstract

Highlights

• •Chromatin immunoprecipitation followed by reChIP (ChIP-reChIP) in Arabidopsis
• •Procedures for transient warm ambient temperature treatment of Arabidopsis seedlings
• •Guidance on chromatin preparation and immunoprecipitation for ChIP-reChIP
• •Recommendations for qPCR primer design, validation, and data processing

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Epigenetic modifications play key roles in regulating transcription by altering chromatin structure and accessibility in response to internal cues or external stimuli. Here, we present a protocol for assessing bivalent histone modification changes through chromatin immunoprecipitation (ChIP)-reChIP assay in response to warm ambient temperature in Arabidopsis. We describe steps to perform transient warm ambient temperature treatment on Arabidopsis seedlings. We then detailed procedures for ChIP-reChIP, which can be applied to investigate various DNA-protein interaction dynamics in different experimental contexts.

Before you begin

This protocol describes how to perform Chromatin Immunoprecipitation followed by re-Chromatin Immunoprecipitation (ChIP-reChIP) assays to investigate the dynamics of bivalent histone modifications in response to elevated ambient temperatures. Bivalent modifications—marked by both activating and repressive histone marks—are particularly relevant for understanding gene regulation under environmental stress, as they may prime genes for rapid response to changing conditions.² By using antibodies specific to these modifications, this protocol enables precise assessment of how temperature changes impact the enrichment of bivalent marks at target genes. Although developed for temperature response studies, this protocol can be easily adapted to explore other environmental stimuli in plants, including hormone signaling and drought response.

Preparation one: Growing Arabidopsis seedlings

Timing: 8 days

Note: The summarized graphic workflow and timeline for preparing Arabidopsis seedlings are depicted in Figure 1.

• 1.
  Prepare 1X Murashige & Skoog (MS) solid medium for Arabidopsis seedlings.

  • a.
    Dissolve 4.8 g MS powder and 10 g sucrose into 1 L ddH₂O by mixing.
  • b.
    Adjust pH to 5.8 by using 1 M KOH solution.
  • c.
    Add 8 g phytoblend agar into 1 L MS solution to reach the final concentration of 8 g/L.
  • d.
    Autoclave MS medium at 121°C for 20 min and pour medium into petri dishes.
  • e.
    Cut cellophane membrane into similar size of petri dishes.
  • f.
    Soak cellophane membrane into water and autoclave.
  • g.
    After the MS medium is solidified, gently lay a piece of cellophane membrane on top of the medium (Figure 2A).

Note: The semi-permeable cellophane membrane allows nutrients and water from the solid MS medium to reach the seedlings while enabling easy removal of seedlings from the medium without disrupting the roots during sampling.³ Alternatively, autoclaved thin filter papers could be used for this purpose. After autoclaving, the cellophane membrane might get winkled. It is critical to keep the membrane as flat as possible against the solid medium to reduce the air gaps (Figure 2A).

• 2.
  Surface-sterilize Arabidopsis seeds.

  • a.
    Prepare 50% (v/v) bleach with 0.1% Triton-X 100. Store at 4°C for future use.
  • b.
    Aliquot the required numbers of seeds of different Arabidopsis genetic backgrounds into 1.5 mL Eppendorf tubes (EP tubes). To ensure effective sterilization, do not fill up more than 1/3 of the tube with seeds.

    Note: If certain mutant plants exhibit reduced growth, such as lhp1-3, it is recommended to increase the initial quantity of seeds to be used as it is important to use a similar quantity of starting material across different genetic backgrounds and treatment conditions in ChIP assays to minimize experimental bias.

  • c.
    Add 1 mL of bleach solution into each tube and rotate for 8 min.

    CRITICAL: Seeds tend to clump together instead of being fully resuspending when rotated, especially when a large quantity of seeds is included in one EP tube. It is important to check potential clumping during the 8-min surface sterilization and flick the tube vigorously to break up any clumps of seeds.

  • d.
    Briefly centrifuge the tubes to pellet the seeds and carefully remove as much bleach solution as possible in a sterile biosafety cabinet.

    Note: From this step onward, the tubes can only be opened in the sterile biosafety cabinet to maintain sterility.

  • e.
    Add 1 mL sterile ddH₂O into the tubes and fully resuspend the seeds.
  • f.
    Short spin the EP tubes and remove ddH₂O.
  • g.
    Repeat Steps e and f at least for four times.

    Note: When dry seeds are soaked in bleach solution or water, their volume gradually increases over time. Therefore, less and less water could be added into the tube each round of washing. It is advisable to increase repeats of washing steps to fully remove any residual bleach.

• 3.
  Sow seeds on solid MS plates.

  • a.
    Use 1 mL pipette to resuspend seeds in top agar or sterilized ddH₂O.

    Note: Top agar is sterile 0.7% (w/v) phytoblend in ddH₂O, which helps uniformly distribute the seeds on the plate.

  • b.
    Spread seeds evenly on top of the cellophane membrane-covered solid MS plates. Additional top agar or water could be added for efficient spreading (Figure 2B).

    Note: At least one pair of plates are required for each genotype (one plate for high ambient temperature of 27°C and another plate for the control at 21°C. Please refer to Section ‘high ambient temperature treatment’ (Step 1 and 2) for detailed experimental settings).

  • c.
    Air-dry the excessive top agar/water in the biosafety cabinet. You may also use pipetting to remove excessive top agar/water.

    Note: Avoid over-drying the plate. Since the green seedlings will be grown under light conditions for five days, it is important to maintain a certain level of moisture within the sealed plate to support proper growth.

  • d.
    Close the petri dish and seal the plate with micropore surgical tape.
  • e.
    Wrap the plates in aluminum foil and stratify the plates at 4°C for at least 3 days in the dark.

Figure 1.

Preparation for ChIP-ReChIP assay

(A) Schematic overview of the plant growth conditions and treatment process. Seeds were surface sterilized and plated, followed by stratification at 4°C for 3–4 days. Seedlings were grown under long-day conditions (21°C, 2 days) to promote germination and then transferred to short-day conditions (21°C, 3 days). Plants were subsequently divided into two groups: control group (21°C, 4 h) and warm ambient temperature treatment group (27°C, 4 h), before sample collection.

Figure 2.

Key steps in ChIP assay

(A and B) Preparing MS plate and sowing Arabidopsis seeds. Lay a sterile cellophane membrane on top of MS solid medium (A) and evenly spread surface-sterilized seeds using top agar (B).

(C–E) Harvesting and cross-linking 5-day-old seedlings. Collect the treatment group and control group seedlings in 50 mL falcon tube (C) and submerge all seedlings in the fixation buffer (D). Place samples in the sealed vacuum chamber for the desired period of time (E).

(F and G) Filtering seedling lysate for nuclear enrichment. Position a cell strainer over a 50 mL falcon tube, then gently pour the sample and Extraction Buffer 1 mixture through the strainer (F). Allow the mixture to filter through, collecting the flow-through in the falcon tube below (G).

(H) Sonicator setting. The bioruptor is set as High-power mode with a cycle of 0.5 min on and 2 min off. The red pointer in the top right dashboard indicates the duration of ‘on’ (sonication), while the green pointer indicates the duration of ‘off’ (rest).

(I) Electrophoresis to show the distribution of DNA fragments of sheared ChIP samples. The main DNA bands across different samples are at around 200 bp.

(J) Schematic workflow of the ChIP-reChIP experiment. Nuclei are first isolated and subjected to sonication to shear chromatin to the desired fragment size/distribution. The first round of immunoprecipitation (ChIP, IP1) is performed, followed by washing and elution to recover chromatin complexes for the second immunoprecipitation (ReChIP, IP2).

Preparation two: Coupling histone modification antibodies to protein G magnetic beads

Timing: 6 h

• 4.
  Prepare Protein G magnetic beads

  • a.
    Make an adequate amount of 0.5% (w/v) BSA in 1X PBS (BSA/PBS) buffer. (e.g., add 200 mg BSA powder into 40 mL 1X PBS)
  • b.
    Pipette 10–20 μL Magnetic Protein G Dynabeads for each sample.

    Note: Any other alternative magnetic beads conjugated with protein A/G could also be used. It is recommended to consult the manufacturer’s product information for the precise volume of magnetic beads required for each ChIP assay, which entails the beads concentration and binding capacity details. For example, the Magnetic Protein G Dynabeads (Invitrogen, Cat#10003D) used in this protocol have a binding capacity of 2.5–3 μg IgG per 10 μL of bead resuspension. In the meantime, the amount of antibody used in the ChIP assay should be considered to determine the appropriate bead volume. In a typical ChIP assay, we use 2 μg of histone modification antibodies, which requires 10 μL of magnetic beads to match this antibody amount. If the efficacy of the histone modification antibody is not ideal, it is advised to test the optimal beads volume and antibody mass while considering the two factors above in a pilot experiment.

  • c.
    Put the tubes on the magnetic rack, wait for 2 min till the supernatant is clear to collect beads, and remove the buffer.
  • d.
    Add 1.5 mL BSA/PBS into collected beads and fully resuspend them after taking them off the magnetic rack.
  • e.
    Put the tubes on the magnetic rack, collect the beads after 2 min till the supernatant is clear, and remove the buffer.
  • f.
    Repeat steps 4d and 4e to wash the Dynabeads three times. Collect the beads with the magnet after the final wash.
• 5.
  Couple antibody to Protein G magnetic beads.

  • a.
    Add 250 μL BSA/PBS to the magnetic beads in each tube.

    Note: Small volume of the magnetic bead-antibody mixture favors more efficient coupling.

  • b.
    Add the preferred antibody to each tube. Use 2 μg antibody for each sample per 10 μL of Magnetic Protein G Dynabeads.

    Note: The concentration of ChIP-grade antibodies varies. It is advisable to check the detailed product information from the manufacturer to optimize antibody usage. Generally, we use 2 μg of histone modification antibodies with 10 μL of magnetic beads (Invitrogen, Cat#10003D) per reaction. In the ChIP-reChIP assay, we refer the antibody used in the first round of IP as antibody A and the other antibody used in the second round of IP as antibody B. In this study, we used anti-H3K4me3 (Active Motif, #39016) for the first round of IP as antibody A, and the anti-H3K27me3 (Active Motif, #61017) was used for the second round of IP as antibody B. Magnetic beads coupled with antibody A and antibody B should be freshly prepared in advance according to the experimental plan.

  • c.
    Incubate the mixture for at least 5 h on a rotating platform at 4°C.
  • d.
    Wash beads 3 times with 1.5 mL of PBS/BSA.
  • e.
    Remove the PBS/BSA.

Preparation three: Design and validation of ChIP-qPCR primers

Timing: 1 h

• 6.Design Control Primers for Assessing ChIP Efficiency.

For the ChIP experiment, it is crucial to verify that the ChIP experiment has functioned correctly before moving on to test our genes of interest through a series of ChIP-qPCR or ChIP-seq library construction. It is advisable to perform ChIP-qPCR on positive and negative control primers with ChIP DNA product. Design and synthesize one or more pairs of qPCR primers that specifically target chromatin regions known to be enriched by the histone modification of interest, as well as regions for negative controls. These primers will be used in ChIP-qPCR to validate the success of the ChIP assays. For example, in the case of H3K27me3, primers targeting FLC, which is a well-studied gene that has high levels of H3K27me3 deposited through the plant life cycle, could serve as the positive control, while primers targeting housekeeping genes could be used as negative control.⁴

• 7.qPCR Primer Design, Efficiency Testing, and Validation for ChIP Experiments.

In order to test whether the transcriptional regulation of the candidate gene results from the chromatin bivalency changes, one of the prerequisites of is that this chromatin region is readily marked by the bivalent histone modifications. It is crucial to analyze the published ChIP-seq datasets of the histone modifications of interest to detect the presence of such epigenetic marks on the candidate gene.

To ensure the accuracy and reliability of histone modification enrichment measurements in our ChIP-qPCR experiments, it is necessary to design qPCR primers at several genomic locations of our genes of interest. It is recommended to design qPCR primers along where the histone modification occurs on the related chromatin regions, as identified by publicly available ChIP-seq datasets (Figure 3A). However, before proceeding with the actual ChIP-qPCR experiments, it is essential to evaluate the efficiency and specificity of these qPCR primers. Primer efficiency significantly impacts the quantification cycle (Ct) values, and suboptimal primers may result in poor amplification, leading to artificially high Ct values, substantially reducing qPCR accuracy. To address this issue, after designing multiple primer pairs for each gene of interest, it is highly recommended to conduct a pilot qPCR experiment to assess their performance. Generally, extracted genomic DNA or DNA input from other ChIP assays could be used as a DNA template in this pilot test. For instance, in the original study by Shao et al., we initially designed three pairs of qPCR primers targeting the GA3ox1 region where both H3K4me3 and H3K27me3 are present (Figure 3A). A pilot qPCR experiment by using the same 100 ng genomic DNA as template revealed that primer pair P2 exhibited poor amplification efficiency when compared to primer pair P1 and P3 as it shows highest Ct value (Figures 3B and 3C). Consequently, we selected primer pair P1, which demonstrated high amplification efficiency and is located near the bivalently marked transcription start site, for subsequent analyses. Additionally, melting curve analysis could also be included to evaluate the specificity of each primer pair, ensuring the amplification of a single, specific product without secondary peaks.

Figure 3.

ChIP-ReChIP assay

(A) ChIP-seq IGV snapshots of H3K27me3 and H3K4me3 enrichment at GA3ox1. Horizontal black lines represent the different amplicons used in ChIP-qPCR and indicate the positions of three designed qPCR primer pairs (P1, P2, P3) within the target region. This figure is adapted from Figure S5C in Shao et al.¹

(B) Summary of the qPCR primers used for ChIP analysis, For each primer pair, forward (F) and reverse (R) primer sequences, amplicon sizes (bp), and average Ct values from technical triplicates in qPCR were listed. The same amount of DNA template (100 ng Arabidopsis genomic DNA) was used for each qPCR reaction.

(C) Primer amplification efficiency measured as 2^-Ct. The bar graph illustrates that P2 exhibits significantly lower efficiency compared to P1 and P3. Error bars represent standard deviation (SD) from technical triplicates and each data point was plotted as a dot.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal histone anti-H3K4me3	Active Motif	Cat#39016; RRID: AB_2687512
Mouse monoclonal histone anti-H3K27me3	Active Motif	Cat#61017; RRID: AB_2614987
Chemicals, peptides, and recombinant proteins
37% formaldehyde	Sigma-Aldrich	Cat#252549
Linsmaier & Skoog with buffer	Caisson Labs	Cat#LSP03-100LT
Phenylmethanesulfonylfluoride (PMSF)	Sigma-Aldrich	Cat#P7626
Proteinase K	New England Biolabs	Cat#P8107S
Dynabeads protein G for immunoprecipitation	Invitrogen	Cat#10003D
Glycine	Research Products International	Cat#G36050-5000.0
Pierce protease inhibitor mini tablets, EDTA-free	Thermo Fisher Scientific	Cat#A32955
Triton X-100	Sigma-Aldrich	Cat#T8787
Phytoblend	Caisson Labs	Cat#PTP01-2.5KG
100 bp DNA ladder	New England Biolabs	Cat#N3231S
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	Cat#71725
Sarkosyl	Thermo Fisher Scientific	Cat#BP234-500
Phenol:chloroform:isoamyl alcohol, 25:24:1 mixture	Bioworld	Cat#41620088-3
Critical commercial assays
Qubit dsDNA HS assay kit	Thermo Fisher Scientific	Cat#Q32851
Other
DynaMag-2 magnet	Thermo Fisher Scientific	Cat#12321D
Qubit fluorometer	Thermo Fisher Scientific	Cat#Q33238
Fisherbrand sterile cell strainers (40 μm)	Fisherbrand	Cat#22363547
Bioruptor	Diagenode	Cat#UCD-200

Materials and equipment

All buffers listed below can be prepared in advance and stored at 4°C. However, PMSF and protease inhibitors should be added fresh and immediately before use.

Extraction buffer 1

Reagent	Final concentration	Stock concentration	Add to 50 mL
Sucrose	0.4 M	2 M	10 mL
Tris-HCl, pH 7.4	10 mM	1 M	0.5 mL
PMSF	1 mM	100 mM	500 μL
Pierce Protease Inhibitor	1 x	100 x	500 μL

Note: Store at 4°C for up to one month. PMSF and Pierce Protease Inhibitor should be freshly added to the buffer prior to use.

Extraction buffer 2

Reagent	Final concentration	Stock concentration	Add to 20 mL
Sucrose	0.25 M	2 M	2.5 mL
Triton X-100	1%	20%	1 mL
Tris-HCl, pH 7.4	10 mM	1 M	0.2 mL
MgCl2	10 mM	1 M	0.2 mL
PMSF	1 mM	100 mM	200 μL
Pierce Protease Inhibitor	1 x	100 x	200 μL

Note: Store at 4°C for up to one month. PMSF and Pierce Protease Inhibitor should be freshly added to the buffer prior to use.

Extraction buffer 3

Reagent	Final concentration	Stock concentration	Add to 20 mL
Sucrose	1.7 M	2 M	17 mL
Triton X-100	0.15%	20%	150 μL
Tris-HCl, pH 7.4	10 mM	1 M	0.2 mL
MgCl2	10 mM	1 M	0.2 mL
PMSF	1 mM	100 mM	200 μL
Pierce Protease Inhibitor	1 x	100 x	200 μL

Note: Store at 4°C for up to one month. PMSF and Pierce Protease Inhibitor should be freshly added to the buffer prior to use.

Nuclei lysis buffer

Reagent	Final concentration	Stock concentration	Add to 20 mL
Tris-HCl, pH 7.4	50 mM	1 M	1 mL
Sarkosyl	0.50%	10%	1 mL
NaCl	100 mM	5 M	0.4 mL
EDTA, pH 8	2 mM	0.5 M	80 μL
PMSF	1 mM	100 mM	200 μL
Pierce Protease Inhibitor	1 x	100 x	200 μL

Note: Store at 4°C for up to one month. PMSF and Pierce Protease Inhibitor should be freshly added to the buffer prior to use.

ChIP dilution buffer

Reagent	Final concentration	Stock concentration	Add to 20 mL
Tris-HCl, pH 7.4	50 mM	1 M	1 mL
Triton X-100	1.25%	20%	1.25 mL
NaCl	100 mM	5 M	0.4 mL
EDTA, pH 8	2 mM	0.5 M	80 μL
PMSF	1 mM	100 mM	200 μL
Pierce Protease Inhibitor	1 x	100 x	200 μL

Note: Store at 4°C for up to one month. PMSF and Pierce Protease Inhibitor should be freshly added to the buffer prior to use.

Low salt wash buffer

Reagent	Final concentration	Stock concentration	Add to 40 mL
Tris-HCl, pH 7.4	50 mM	1 M	2 mL
Triton X-100	0.50%	20%	1 mL
NaCl	150 mM	5 M	1.2 mL
EDTA, pH 8	2 mM	0.5 M	160 μL

Note: Store at 4°C for up to one month.

High salt wash buffer

Reagent	Final concentration	Stock concentration	Add to 40 mL
Tris-HCl, pH 7.4	50 mM	1 M	2 mL
Triton X-100	0.5%	20%	1 mL
NaCl	500 mM	5 M	4 mL
EDTA, pH 8	2 mM	5 M	160 μL

Note: Store at 4°C for up to one month.

LiCl wash buffer

Reagent	Final concentration	Stock concentration	Add to 40 mL
Tris-HCl, pH 7.4	50 mM	1 M	2 mL
Sodium Deoxycholate	1%	10%	4 mL
LiCl	250 mM	5 M	2 mL
EDTA, pH 8	2 mM	5 M	160 μL
Igepal CA-630	1%	10%	4 mL

Note: Store at 4°C for up to one month.

Final wash buffer

Reagent	Final concentration	Stock concentration	Add to 40 mL
Tris-HCl, pH 7.4	50 mM	1 M	2 mL
NaCl	50 mM	5 M	0.4 mL
EDTA, pH 8	2 mM	0.5 M	160 μL

Note: Store at 4°C for up to one month.

Elution buffer

Reagent	Final concentration	Stock concentration	Add to 5 mL
Tris-HCl, pH 8	50 mM	1 M	250 μL
EDTA, pH 8	2 mM	0.5 M	160 μL
SDS	1%	10%	500 μL

Note: Store at 4°C for up to one month.

ReChIP elution buffer

Reagent	Final concentration	Stock concentration	Add to 5 mL
Tris-HCl, pH 8	50 mM	1 M	250 μL
EDTA, pH 8	2 mM	0.5 M	200 μL
SDS	1%	10%	500 μL
DTT	10 mM	100 mM	500 μL

Note: Should be freshly prepared.

Step-by-step method details

High ambient temperature treatment

Timing: 5 days

This section outlines adjustable high-temperature treatments and consistent light conditions for controlled comparisons.

This protocol outlines a transient high ambient temperature treatment for 4 h in 5-day-old seedlings grown under short-day conditions (8 h light, 16 h dark). Alternatively, longer exposure to high ambient temperatures, ranging from 12 h to 5 days, can be applied based on the specific experimental objective; long-day or continuous light conditions may also be used.

Note: If the gene of interest is regulated by the circadian clock or is sensitive to light exposure, continuous light conditions are recommended to minimize variation. Two critical aspects of high-temperature treatments are (1) ensuring that both the control and treatment groups are sampled at the same time point and (2) maintaining consistent light intensity settings in both growth chambers of regular ambient temperature (21°C) and warm ambient temperature (27°C), as light signaling is intricately linked with the warm temperature response.

• 1.
  Grow Arabidopsis Seedlings at Regular Ambient Temperature.

  • a.
    Remove the aluminum foil and place the MS plates into a 21°C growth chamber (100 μmol m⁻² s⁻¹ light intensity with a long day setting of 16 h light and 8 h dark) to promote germination for 2 days.
  • b.
    Move the plates with germinated seeds into short day growth chamber (100 μmol m⁻² s⁻¹ light intensity with a short day setting of 8 h light and 16 h dark) for 3 days.
• 2.
  High Ambient Temperature Treatment and Sample Collection.

  • a.
    Move one group of plates into a 27°C chamber while the other group remains in the original 21°C chamber for 4 h.

    Note: The starting time point (ZT time) for high ambient temperature treatment may potentially influence experimental outcomes. Therefore, it is strongly recommended to initiate the treatment at the same ZT time for all biological replicates to ensure consistency and reproducibility in the results.

  • b.
    Collect all seedlings and put them into 50 mL falcon tubes (Figure 2C). Immediately move on to the next step for crosslinking.

Fixation: Cross-link chromatin

Timing: 40 min

This step cross-links histones and DNA in seedlings to preserve chromatin states for ChIP, with optional dual cross-linking for complex interactions.

The goal of this step is to use formaldehyde to cross-link histones and DNA in Arabidopsis seedlings for the following ChIP assays. This step enables rapid fixation of chromatin dynamics immediately after sampling, while minimizing the risk of over-fixation. For studying chromatin-associated proteins that do not directly bind to DNA, dual cross-linking methods may be applied, which can be achieved by using longer-arm cross-linkers, such as ethylene glycol bis-(succinimidyl succinate) (EGS), to capture more complex protein-DNA or protein-protein interactions.⁵ It is recommended to perform the entire procedure at 4°C and prechill every buffer used in this step.

• 3.
  Fixation.

  • a.
    Prepare a 10 mM HEPES-NaOH buffer (pH = 7.4) by diluting from stock HEPES-NaOH solution (100 mM, pH = 7.4).
  • b.
    Freshly prepare an adequate volume of Fixation Buffer which contains 1% (0.3 M) of formaldehyde in 10 mM HEPES-NaOH buffer (pH = 7.4).

Note: For each sample of green seedlings weighing around 1–2 g, 20–30 mL of Fixation Buffer should be sufficient.

• 4.
  Vacuum infiltration.

  • a.
    Submerge seedlings into Fixation Buffer in 50 mL falcon tube with vacuum infiltration for 5 min (Figures 2D and 2E).

    Note: It is important to ensure that the vacuum jar is fully sealed. This can be checked by lifting the entire vacuum jar by holding the lid. If the jar is completely sealed, the base will remain attached.

  • b.
    Gently release the vacuum and thoroughly mix the seedlings with Fixation Buffer by either flipping the Falcon tube or stirring the solution.

    Note: After vacuuming, the seedlings may float above the Fixation Buffer, and bubbles may also form around the seedlings. This step ensures that these bubbles are fully removed and that the samples are submerged in the Fixation Buffer for proper fixation.

  • c.
    Start the vacuum infiltration for 10 min and repeat Step 4a-4b.
  • d.
    Start the vacuum infiltration for another 5 min and repeat Step 4a-4b.

    Note: The full vacuum infiltration procedure is vacuuming for 5 + 10 + 5 mins with Step 4b in the interval.

• 5.
  Quenching Fixation.

  • a.
    Add 670 μL 2 M Glycine (dissolved in 10 mM HEPES-NaOH, pH 7.4) to every 10 mL of Fixation Buffer to reach the final concentration of 125 mM to quench the formaldehyde.
  • b.
    Mix well the solution-seedling mixture.
  • c.
    Continue vacuum infiltration for an additional 5 min.
• 6.
  Washing and Storage.

  • a.
    Rinse seedlings with 10 mM HEPES-NaOH, pH 7.4.
  • b.
    Use a paper towel to dry up the seedlings.
  • c.
    Wrap the seedlings in foil or put into a 50 mL falcon tube, snap freeze in liquid nitrogen, and store at – 80°C.

Note: Frozen plant samples should be used within two months to avoid protein and DNA degradation for the optimal assay performance.

Nuclei isolation

Timing: 2 h

This step ensures clean nuclei isolation from frozen Arabidopsis seedlings. The removal of non-nuclear contents will efficiently enrich chromatin and reduce non-specific binding during the IP process.

• 7.
  Cell Lysis.

  • a.
    Grind the frozen seedlings in liquid nitrogen to a fine powder by using a mortar and pestle.
  • b.
    Add 5 mL of Extraction Buffer 1 for every 1 g of ground powders in a 50 mL falcon tube for each sample.

    Note: The minimum starting material weight varies, depending on the antibody efficacy. It is advised to perform a pilot experiment to determine the minimum sample requirement for a specific ChIP assay. Generally, for ChIP assays targeting histones, a few of the most abundant proteins bound to chromatin, around 1 g of starting material is suggested. But it is critical to use the same weight of starting material across different samples to reduce variation.

  • c.
    Incubate for 10 min on a rotating platform at 4°C to facilitate cell lysis.
• 8.
  Non-nuclear Contents Removal and Nuclei Enrichment.

  • a.
    Filter homogenized samples through cell strainer (40 μm nylon mesh, Fisher brand, Cat No. 22363547) and collect the flow-through (Figures 2F and 2G).

    Note: Alternatively, you can filter the solution through one single layer of pre-wet Miracloth twice into a clean 50 mL falcon tube. This step could be carried in an ice box or in the cold room.

  • b.
    Spin the filtered solution for 20 min at 2880 x g in a centrifuge at 4°C.
  • c.
    Gently remove supernatant and thoroughly resuspend the pellet in 1 mL Extraction Buffer 2. Transfer the solution to 1.5 mL Eppendorf tube.
  • d.
    Centrifuge at 12,000 × g for 10 min at 4°C.

    Note: A tight white pellet (nuclei and junk) should be visible, along with an overlay of chlorophyll.

  • e.
    Remove supernatant and thoroughly resuspend pellets in 500 μL of Extraction Buffer 3.

    Note: This resuspension can be challenging; take care and try to avoid foaming.

  • f.
    Add 500 μL of Extraction Buffer 3 in a clean tube to establish the density gradient.
  • g.
    Take the 500 μL solution (resuspended pellet) from the last step and carefully layer it on top of the clean 500 μL of Extraction Buffer 3.
  • h.
    Spin for 1 h at 16,000 × g at 4°C.

    Note: It is recommended to perform nuclei counting using a hemocytometer or an automated cell counter to estimate nuclei yields across different samples for input normalization and to assess nuclei integrity. However, extracted nuclei should be processed promptly, as prolonged delays before proceeding with the sonication steps may lead to DNA-protein complex degradation.

Nuclei lysis and DNA sonication

Timing: 1.5 h

• 9.
  Lysing Enriched Nuclei.

  • a.
    Discard the supernatant of Extraction Buffer 3 from the last step.
  • b.
    Thoroughly resuspend chromatin pellet in 300 μL of Nuclei Lysis Buffer.
  • c.
    Transfer 300 μL resuspended chromatin to the new tube to reduce contamination.
  • d.
    Gently rotate the sample at 4°C for 20 min for thorough nuclei lysis.
• 10.
  Shearing chromatins by sonication.

  • a.
    Set the Bioruptor setting at High mode with 0.5 min on and 2 min off (Figure 2H).

    Note: The power of sonicators varies on different models. Given the optimal sheared chromatin size around 200–300 bp, it is recommended to test the sonicator setting prior to the actual ChIP assays.

  • b.
    Sonicate chromatin solution in a Bioruptor for 11 min as one repeat.

    Note: One sonication repeat of 11 mins contains 5 cycles of 0.5 min on and 2 min off.

  • c.
    Put samples on ice for 1–2 min to cool down.

    Note: If the water in the Bioruptor becomes warm, it is recommended to change it to ice water to stabilize the samples.

  • d.
    Repeat the sonication for 5 times in total (5 × 11 mins).

    Note: It is recommended to perform a pilot experiment or run sheared chromatin samples on a DNA gel at this stage to determine the optimal sonication duration and prevent insufficient or excessive shearing. However, keep in mind that electrophoresis of sheared chromatin immediately after sonication may not accurately reflect the actual DNA fragment size, as DNA and chromatin-bound proteins remain covalently crosslinked. This step should be used as a rough reference rather than a precise measurement of running de-crosslinked DNA (Figure 2I).

• 11.
  Collecting sheared soluble chromatin fragment.

  • a.
    Centrifuge the sample for 10 min at max speed in centrifuge at 4°C.
  • b.
    Transfer the supernatant to a new tube and spin again for 10 min at 4°C to remove the additional debris.
  • c.
    Transfer 300 μL supernatant into low-binding Eppendorf tubes.

Note: Alternatively, the supernatants can be stored at −80°C up to two months, if not proceeding to IP.

Immunoprecipitation and washing

Timing: 12–16 h + 2 h

This step involves the incubation of sheared chromatin fragments with antibody-coupled protein G magnetic beads for immunoprecipitation, followed by efficient washing after the IP step. The duration of immunoprecipitation incubation can be reduced if the antibody used has high efficacy to avoid degradation. As histone immunoprecipitations tend to produce a high background, a more rigorous washing procedure is recommended in this protocol to minimize non-specific binding. However, for ChIP assays targeting other chromatin-associated proteins, such as transcription factors, the washing time can be shortened to reduce chromatin loss, while still ensuring efficient immunoprecipitation.

• 12.
  Immunoprecipitation of Chromatin Fragments.

  • a.
    Dilute the 300 μL soluble chromatin solution with 4 volumes (1200 μL) of ChIP Dilution Buffer.
  • b.
    Save 60 μL diluted samples as input chromatin controls (4% input) and store the input for each sample at – 80°C.

    CRITICAL: It is essential to save a small aliquot of the diluted chromatin solution from each sample at this step. These input samples are crucial for normalizing the amount of DNA input in subsequent ChIP-qPCR assays or ChIP-seq analyses, enabling accurate comparison of enrichment levels.

  • c.
    Remove the supernatant of antibody-coupled magnetic beads from Preparation Two after magnetic rack collection and add the diluted samples into each tube.
  • d.
    Bind chromatin fragments to magnetic beads coupled with antibody A at 4°C for 12–16 h (overnight) with gentle rotation.
  • e.
    Collect beads on a magnetic rack after 2-min’s wait till the supernatant is clear and discard the supernatant.
• 13.
  Washing Magnetic Beads.

  • a.
    Prepare and prechill fresh Low Salt Buffer, High Salt Buffer, and Final Wash Buffer.
  • b.
    Resuspend magnetic beads at 4°C with 1 mL of the Low Salt Buffer by flipping.
  • c.
    Put in ice for 1 min.
  • d.
    Collect the beads on a magnetic rack, wait for 2 min until the supernatant is clear, and resuspend in 1 mL of the Low Salt Buffer.
  • e.
    Rotate for 5 min in the cold room.

    Note: Steps 13b and 13c are quick washing of magnetic beads, while steps 13d and 13e are slow washing of magnetic beads.

  • f.
    Use High Salt Buffer to perform 1 min quick wash and 5 min slow wash for the magnetic beads.
  • g.
    Use LiCl Buffer to perform 1 min quick wash and 5 min slow wash for the magnetic beads.
  • h.
    Use Final Wash buffer to perform a 1 min quick wash followed by a 5 min slow wash of the magnetic beads.

Chromatin elution and reChIP

Timing: 1 h + 12–16 h

• 14.
  Elution of Chromatin Fragments from Magnetic Beads.

  • a.
    Resuspend collected beads in 100 μL of ReChIP Elution Buffer.
  • b.
    Incubate tubes for 15 min at 37°C with on a shaker with 120 rpm.

    Note: Although a lower-temperature elution is used, 37°C is sufficient to facilitate the dissociation of IP products from magnetic beads and to heat-deactivate DTT, while maintaining the stability of the DNA-chromatin complex.⁶

  • c.
    Do a second elute with another 100 μL ReChIP Elution Buffer at 37°C for 15 min.
  • d.
    Combine eluents (200 μL in total).
• 15.
  ReChIP.

  • a.
    Aliquot the eluted chromatin fragment from Step 14d equally into four low-binding Eppendorf tubes (50 μL per tube).
  • b.
    Add 150 μL of Elution Buffer to each of the four tubes, bringing the total volume in each tube to 200 μL.
  • c.
    Set aside one tube (IP1: Antibody A), along with the input sample from Step 12b, to proceed directly through Steps 16–18.

    Note: IP1 refers to the first round of IP and IP2 refers to the second round of IP. IP1: Antibody A refers to the first round of IP using antibody A coupled magnetic beads.

  • d.
    For the remaining three tubes, add 300 μL of ChIP Dilution Buffer (total volume 500 μL) and:
    Tube 1 (reChIP IP2: Antibody B): Add magnetic beads coupled with antibody B to this tube for re-ChIP and proceed with Steps 12–13, followed by Steps 16–18.
    Tube 2 (reChIP IP2: Antibody A): Add magnetic beads coupled with antibody A to this tube for re-ChIP and proceed with Steps 12–13, followed by Steps 16–18.
    Tube 3 (Mock reChIP): Perform a mock reChIP using the eluted chromatin fragment without adding any antibody (add equilibrated and blocked magnetic beads from Preparation Two 4f). Process this tube by following Steps 12–13, and then continue with Steps 16–18.

    Note: After dilution, the final SDS concentration during the overnight IP is 0.4%, which may be incompatible with certain antibodies or make it difficult to detect low-abundance histone modifications. If encountering such issues, we recommend maintaining the final SDS concentration between 0.1–0.2% for milder IP conditions. Additionally, ion-exchange columns could be used for DTT removal in Step 15d as an equivalent approach. From our experiences, desalting eluted ChIP samples using ion-exchange columns can reduce chromatin recovery and may not be ideal for small sample volumes. Alternatively, diluting eluted ChIP samples to lower the DTT concentration works effectively for the second IP.⁶

Chromatin elution and de-crosslinking

Timing: 1 h + 6–16 h

• 16.
  Final Chromatin Elution.

  • a.
    Resuspend collected beads in 100 μL of Elution Buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS).
  • b.
    Incubate tubes for 15 min at 65°C with on a shaker with 120 rpm.
  • c.
    Do a second elute with another 100 μL Elution Buffer at 65°C for 15 min.
  • d.
    Combine eluents (200 μL in total).
  • e.
    Add 140 μL Elution Buffer to the chromatin input control tube from Step 12b.
• 17.
  De-crosslinking.

  • a.
    Add 5 M NaCl solution in each tube to achieve 0.2 M NaCl.

    Note: From this step, all samples including IP samples and Input samples are subject to the same procedure.

  • b.
    Incubate overnight (ranging from 6 to 16 h) at 65°C.
  • c.
    Add 1 μL Proteinase K (800 units/mL) to each tube.
  • d.
    Incubate for 2 h at 55°C with shaking.

DNA purification

Timing: 1 h + 3–16 h

• 18.
  ChIP DNA Extraction.

  • a.
    Add 200 μL (equal amount of DNA solution) phenol/chloroform/isoamyl alcohol (25:24:1, pH = 8.0) to DNA solution.

    Note: Use the lower phase of the phenol/chloroform/isoamyl alcohol solution, as the mixture typically separates into two layers in the original container. The top layer is only for sealing phenol/chloroform/isoamyl alcohol and should not be used.

  • b.
    Vigorously vortex the tube for 30 s.
  • c.
    Centrifuge at 12000 x g for 10 min.
  • d.
    Recover the upper phase to a new tube.

    Note: The volume of the upper phase will be reduced to around 180–190 μL.

• 19.
  ChIP DNA Precipitation.

  • a.
    Add 8 μL of 5 M NaCl and 2.5 times of sample volume of 100% ethanol (500 μL) into each sample.

    Note: The final concentration of NaCl shall be 0.2 M (4 μL of 5 M NaCl / 100 μL sample).

  • b.
    Incubate at −80°C for at least 3 h or overnight (approximately 16 h).
  • c.
    Centrifuge for 30 min at max speed in centrifuge at 4°C.
  • d.
    Wash the pellet with 1 mL of 75% ethanol. Repeat the wash once.
  • e.
    Air-dry the DNA pellet in the fume hood.
  • f.
    Resuspend the DNA pellet in 50 μL ddH₂O.
  • g.
    Quantify DNA concentration with Qubit fluorometer or Nanodrop.

    Note: If the DNA pellet is over-dried, it might take longer for DNA to be dissolved in the ddH₂O. Please make sure that DNA is dissolved before proceeding with the next steps.

Expected outcomes

Sheared DNA fragments

For optimal ChIP analysis, most of sheared DNA fragments should fall within the 200–300 bp range (Figure 2I). If ChIP-qPCR is used as downstream experimental approach, slightly longer DNA fragments (up to 500 bp) are still acceptable, as the typical amplicon size for ChIP-qPCR is around 100–200 bp. However, for ChIP sequencing, it is crucial to strictly control the fragmented DNA size within the 200–300 bp range to ensure optimal mapping accuracy during deep sequencing analysis.

ChIP enrichment of specific genomic regions

If the ChIP assay is successful, we should observe the preferential enrichment of specific genomic regions marked by the histone modification of interest. As described in Preparation Three, using positive control primers to evaluate ChIP efficiency through ChIP-qPCR should show enriched ChIP signals relative to the input. Once confirmed, we can examine our genes of interest to determine whether their bivalent histone modifications respond to high ambient temperature, or any other treatment condition. For example, to evaluate the H3K4me3-H3K27me3 bivalency dynamics at the GA3ox1 gene locus (Figure 3), we performed qPCR on DNA samples from the following groups: input, IgG, IP1 (H3K4me3), IP2 (H3K4me3), and IP2 (H3K27me3). ChIP-qPCR data were normalized by calculating ChIP enrichment for the IP and IgG negative control relative to the input in the 27°C treatment group compared to the 21°C control group (Figure 4A). Specifically, the percentage relative to input was determined using the formula:

$$\text{Relative}\text{to}\text{Input}=100\times 2^{(\text{Ct}\left(\text{input}\right)-4.64-\text{Ct}\left(\text{IP}\right))}$$

Figure 4.

Analysis of ChIP-reChIP qPCR Data

(A) Summary of ChIP-qPCR raw data obtained in ChIP-reChIP assay. Average Ct values from ChIP-qPCR for the first (IP1) and second (IP2) immunoprecipitation steps under 21°C and 27°C conditions were listed for input, IgG control, IP1 (H3K4me3), IP2 (H3K4me3), and IP2 (H3K27me3) at the GA3ox1 locus.

(B) Sequential ChIP-qPCR analysis of H3K4me3 and H3K27me3 enrichment at the GA3ox1 under 21°C and 27°C conditions. The bar graph shows the relative ChIP enrichment (% of input) for each IP step as indicated in the figure. The ratio of IP2/IP1 (IP2: H3K27me3 / IP1: H3K4me3) is calculated to indicate the reduced H3K27me3 enrichment levels and the increased H3K4me3 levels in Arabidopsis wild-type plants in response to warm ambient temperature. Error bars represent standard deviation (SD) from quadruplicates and each data point is calculated as a dot. The raw data used in this figure were derived from Figure S5H in Shao et al.¹

Since 4% of total chromatin was used as input, the term Ct(input) - 4.64 adjusts the input to 100% (log₂0.04 = −4.64). Additionally, the ratio of IP2 (H3K27me3) to IP1 (H3K4me3) was calculated to illustrate relative changes in H3K4me3-H3K27me3 bivalency between the two temperature conditions at the GA3ox1 locus in wild-type Arabidopsis (Figure 4B).

Alternatively, the ChIP product from this assay can be used for deep sequencing library preparation to identify novel bivalent chromatin regions or to assess genome-wide bivalency alternation in response to the treatment of interest. After mapping and normalization, we are able to compare the levels of histone modifications between different samples and treatments. We recommend following the manufacturer’s protocol for detailed deep sequencing library preparation (NEBNext Ultra II DNA Library Prep Kit for Illumina, Cat#E7645S).

Limitations

The development and success of a ChIP-reChIP assay largely depend on the antibody’s ability to specifically recognize the histone modification of interest. If a noncanonical histone modification is being studied, it might be challenging to find a commercially available ChIP-grade antibody for the assay.

The method described here uses formaldehyde as the cross-linking agent, which cross-links proteins directly associated with DNA, such as histones in this case. For studies involving chromatin-associated proteins that do not directly bind to DNA, alternative cross-linkers should be used in the ChIP assay to capture these interactions effectively.⁵

Troubleshooting

Problem 1

Low seed germination rate.

Potential solution

Several factors can contribute to low germination rate in Arabidopsis green seedlings. From the technical perspective, prolonged incubation of seeds with bleach solution, insufficient washing to remove residual bleach, and the over-drying of solid MS plates will all drastically reduce the seeds’ ability to germinate. From the biological perspective, low seed quality (poor growth condition of parental plants), sowing fresh seeds too soon (unfinished post-embryonic dormancy), and insufficient stratification time (no cold treatment or the treatment time is shorter than two days) would have a negative impact on seedling growth. Additionally, some mutant plants tend to have slow timing for germination, which needs to be taken into consideration to achieve equal sampling whether to increase seed amounts for mutants that do not germinate well. Therefore, it is important to check the seed condition before large-batch material preparation and treatment.

Problem 2

Suboptimal ChIP DNA size distribution.

Potential solution

The power of sonicators can vary among different sonicator models. It is recommended to run a pilot test to see under which setting the appropriate shearing could be archived. The amount of sample into sonication processing should also be taken into consideration. Using too little starting material can lead to over-shearing, while excessive starting material may result in insufficient shearing. Although the sheared DNA fragments will always be in a range, using similar amounts of starting material for consistent results is highly advisable.

Problem 3

Low DNA recovery.

Potential solution

If both the input DNA and the IP product show low DNA concentrations despite using adequate starting materials (empirical input DNA concentration: 10–50 ng/μL; empirical IP product DNA concentration: 0.1–5 ng/μL from 2 g of samples), it could be due to insufficient tissue lysis, poor nuclei enrichment, or incomplete DNA precipitation. It is recommended to check the yield of nuclei (a visible nucleus pellet should be seen around 50–100 μL from 2 g of green seedlings). Additionally, increasing the DNA precipitation time to overnight and extending the centrifugation duration at 4°C may help maximize the DNA recovery.

If only the IP product has low DNA concentrations while the input DNA does not, several technical issues could lead to this situation. First, inadequate cross-linking may reduce the amount of chromatin that can be immunoprecipitated. Since the cross-linking agent formaldehyde is unstable, it is advisable to use freshly prepared cross-linking buffers and ensure the quality of the formaldehyde used in this assay. Second, antibody efficacy is a critical factor that influences IP efficiency. If low ChIP enrichment is observed, antibody performance can be verified via immunoblotting to confirm whether the antibody effectively recognizes the histone modification of interest. Furthermore, the quantity of antibody used in the ChIP assay should be tested and optimized. Manufacturers often indicate whether an antibody is ChIP-grade in the product manual, so using antibodies specifically validated for ChIP is recommended to ensure optimal results.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hong Qiao (hqiao@austin.utexas.edu).

Technical contact

Further information about technical support should be directed to Zhengyao Shao (zhs035@health.ucsd.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate new data or code.

Acknowledgments

We thank F. Zhang and L. Wang for the initial experiment setup. The graphical abstract was created in BioRender. This work was supported by grants from the National Institutes of Health to H.Q. (NIH-2R01 GM115879).

Author contributions

Conceptualization, Z.S. and H.Q.; investigation, Z.S. and H.Q.; writing – original draft, Z.S.; writing – review and editing, H.Q.; funding acquisition, H.Q.

Declaration of interests

The authors declare no competing interests.