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Spray bottle TurnNSpray 500 ml (R&L Slaughter, catalog number: 215-2962)
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Corning 50 ml centrifuge tubes (Sigma-Aldrich, catalog number: CLS430829-500E)
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Elastic rubber bands
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Paper towel
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Aluminium Foil
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Funnel (6 cm diameter)
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2 ml DNA low binding (LoBind) tubes (Eppendorf, catalog number: 0030108078)
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1.5 ml DNA low binding (LoBind) tubes (Eppendorf, catalog number: 022431021)
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Arabidopsis thaliana
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Indole-3-acetic acid (Sigma-Aldrich, catalog number: I5148)
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Silwet L-77 (De Sangosse Ltd., catalog number: 0640)
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Miracloth (MERK, catalog number: 475855)
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Liquid nitrogen
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10x Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: 11666789001)
-
Formaldehyde (Sigma-Aldrich, catalog number: F8775)
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Glycine (Thermo Fisher Scientific, catalog number: G/0800/60)
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Sterile water
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Succrose (Thermo Fisher Scientific, catalog number: S/8600/60)
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Ficoll 400 (Sigma-Aldrich, catalog number: F8636)
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Dextran from Leuconostoc spp. (Sigma-Aldrich, catalog number: 31389)
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HEPES (Sigma-Aldrich, catalog number: H4034)
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Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266)
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Triton X-100 (Sigma-Aldrich, catalog number: T9284)
-
DL-Dithiothreitol (DTT; Sigma-Aldrich, catalog number: D0632)
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cOmplete EDTA-free protease inhibitor cocktail (Roche Molecular Systems, catalog number: 4693132001)
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Tris(hydroxymethyl)aminomethane (TRIS; Sigmal-Aldrich, catalog number: B2005)
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Ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, catalog number: E6758)
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20% Sodium Dodecyl Sulfate (SDS) solution (Severn Biotech; catalog number: 20-4002-05)
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Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S7653)
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Dynabeads Protein A (Invitrogen, catalog number: 10001D)
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Anti-H3 Antibody (Rabbit polyclonal Antibody; Abcam, catalog number: ab1791)
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Anti-H3K27ac Antibody (Rabbit polyclonal Antibody; Abcam, catalog number: ab4729)
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Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761)
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Proteinase K (Roche Molecular Systems, catalog number: 40693800)
-
Phenol:Chloroform:Isoamyl alcohol (25:24:1) (Sigma-Aldrich, catalog number: 77617)
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Sodium acetate (NaAc) (Sigma-Aldrich, catalog number: S7670)
-
Ethanol absolute (VWR Chemicals, catalog number: 20821.330)
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LightCycler 480 Multiwell Plate 96, white (Roche Molecular Systems, catalog number: 04729692001)
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LightCycler® 480 Sealing Foil (Roche Molecular Systems, catalog number: 04729757001)
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Oligo-nucleotide primers (Integrated DNA Technologies)
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SYBR Green JumpStart Taq Ready Mix (Sigma-Aldrich, catalog number: S4438)
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RNase A (Thermo Fisher Scientific, catalog number: EN0531)
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Agarose (Melford Biolaboratories Ltd., catalog number: A20080-1000.0)
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100 bp DNA ladder (New England Biolabs Ltd., catalog number: N3221S)
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Stock Solutions (see Recipes)
2 M Glycine
0.5 M HEPES KOH pH 7.4
1 M MgCl2
1 M Tris-HCl pH 8
0.5 M EDTA
5 M NaCl
3 M NaAc
1 M DTT
20% (v/v) Triton X-100
10% (v/v) SDS
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ChIP Buffers (see Recipes)
Honda Buffer
Nuclei Lysis Buffer
ChIP Dilution Buffer
Low Salt Wash Buffer
High Salt Wash Buffer
TE Buffer
Elution Buffer
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Tweezers (DUMONT BIOLOGICAL TYPE 5 SS-1, TAAB Laboratories Equipment, catalog number: T083)
-
Glass beaker 100 ml
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Mortar and pistil
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Bel-Art Space Saver Vacuum Desiccator (Thermo Fisher Scientific, catalog number: 11852732)
-
Vacuum rump (Edwards, model: IEC34-1)
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Tube rotor (Stuart Scientific, model: SB1)
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Centrifuge (Eppendorf, model: 5810R)
-
Swing-bucket rotor with 50 ml tube adaptors (Eppendorf, model: A-4-81)
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Fixed-angle rotor for 48 x 1.5/2.0 ml tubes (Eppendorf, model: FA-45-48-11)
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Sonicator (Diagenode, Model Bioruptor Plus)
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Magnetic separator rack (GE Healthcare, MagRack6, catalog number 28-9489-64)
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Thermomixer (Eppendorf, Model Eppendorf ThermoMixer C)
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CFX96 touch real-time PCR detection system (Bio-Rad)
-
Vacuum concentrator (Eppendorf, model: Concentator Plus)
-
Gel electrophoresis system (Thermo Electric Corporation, model: CSSU1214)
-
Power pack (Kikusui Electronics Corporation, model: PAB 350-0.2)
-
Indole-3-acetic acid (IAA) is insoluble in water and needs to be freshly prepared in ethanol to a stock concentration of 100 mM. Mock treatment has to contain the same amount of ethanol as the auxin treatment. When treating the plants, make sure that you spray equally with treatment and mock solution. Treatment of ~100 Arabidopsis plants requires ~100 ml solution.
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We only harvested inflorescence tissue that contained closed flower buds. Flowers that were opened or young siliques were removed.
-
To obtain 3 g sample inflorescences of ~100 plants need to be harvested. We recommend to collect one pair of biological replicates (one replicate per genotype, mock and IAA-treatment) at the same time and to repeat collection three times to obtain three replicates for an experiment. We recommend having two people collecting to reduce the time.
-
If including more than one genotype or treatment, it is advised to use one glass beaker per treatment and genotype to prevent mixing of samples.
-
Crosslinking time may need optimisation depending on protein and tissue. The optimal crosslink time crosslinks your protein of interest to the DNA but does not block the epitope. This can be tested by Western blotting.
-
Do not start timing the 5 min before the first bubbles emerge.
-
This can be done with a 100 μl pipette. We recommend cutting the pipette tip to prevent damaging the nuclei.
-
Sonication conditions can differ between sonicators and depend on samples. Generally four parameters are essential for the efficiency of the DNA fragmentation: 1) the temperature of the sample (we recommend to maintain a temperature of 4 °C throughout the sonication), 2) the concentration of formaldehyde used for crosslinking (the higher the concentration leads increases the sonication time), 3) the volume in which sonication is performed (we recommend not to sonicate more than 250 μl of lysate in a 1.5 ml tube), 4) the concentration of SDS in the lysis buffer (more SDS leads to easier lysis during sonication).
-
Sonication test:
Once your sonication conditions are well established, you can skip this step. Bring sonication and non-sonication aliquots to 100 μl volume with elution buffer.
-
Add 1 μl of 10 mg/ml RNase A, mix well and incubate for 1 h at 37 °C.
-
Add 100 μl of Phenol:Chloroform:Isoamyl alcohol (25:24:1), vortex for 30 s and centrifuge 4,000 x g for 10 min at room temperature.
-
Transfer the aqueous phase to a fresh 1.5 ml Lobind tube and add 0.1 volumes (10 μl) of 3 M NaAc (pH 5.5) and 2 volumes (200 μl) of 100% EtOH. Vortex and incubate for 1.5h at -20 °C.
-
Centrifuge for 4,000 x g for 20 min at 4 °C.
-
Remove the supernatant and dry the pellet for 10 min in vacuum concentrator (65 °C).
-
Resuspend the pellet in 10 μl water.
-
Run samples on a 1.5% Agarose gel.
-
Visualise the gel. The DNA should be visible as a smear between 100 bp and 500 bp (Figure 1). The smaller the DNA is fragmented the higher the resolution of the experiment.

Figure 1. Sonication test. Marker (NEB 100 bp ladder); Sample showing the bulk of DNA fragments range from 100-500 bp.
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The volume of water depends on the number of qPCR reactions that will be run (3 μl DNA per reaction).
-
Primers should be designed to amplify fragments (the ideal fragment length is ~100 bp) with regular spacing across the whole gene locus of interest. The resolution of the experiments increases with increasing amplicon density. Before use, all qPCR primers should be tested for their efficiency and specificity. The amplification efficiency of primers can be tested using a genomic DNA dilution series (1/4 to 1/4096) (Figure 2A). The specificity of primers was confirmed by analysing the melting curves (65 °C to 95 °C) (Figure 2B). qPCR reactions should be prepared and performed as described in the experimental procedures (G1-3).

Figure 2. Example a suitable qPCR primer. A. Amplification efficiency of a suitable primer should show a correlation between amplification and dilution (R2) of 0.95-1 and a primer efficiency (10-1/slope) of 2 ± 0.1. B. Melt peak indicating high specificity of the primer pair.
We are grateful to Yang Dong and Emilie Knight for critical comments on the protocol. We also express our gratitude to Caroline Dean, Hongchun Yang and Julia Qüesta for their helpful advice throughout the development of this protocol. This protocol was adapted from
Kuhn et al., 2020
. This work was supported by grant BB/S002901/1 to L.Ø., the Norwich Research Park Biosciences Doctoral Training Partnership [grant number BB/M011216/1 to A.K.] and by the Institute Strategic Programme grant (BB/P013511/1) to the John Innes Centre all from the UKRI Biotechnological and Biological Sciences Research Council.
The authors declare no competing interests.