Universal Plant ChIP-seq kit
Cat. No. C01010152

USER GUIDE
Version 3

02_2024

Please read this manual carefully
before starting your experimentContents
Kit materials ............................................................................................................... 4
Required Materials Not Provided ............................................................................... 6
Remarks before starting ............................................................................................. 7
SHORT PROTOCOL .................................................................................................... 12
DETAILED PROTOCOL ............................................................................................... 18
STEP 1. Crosslinking of plant tissue ................................................................. 19
STEP 2. Chromatin extraction from plant tissue and shearing ......................... 21
STEP 3. Magnetic immunoprecipitation .......................................................... 24
STEP 4. Elution, de-crosslinking and DNA isolation using IPure Kit v2 .............. 26
STEP 5. qPCR analysis and results ................................................................... 28
ADDITIONNAL PROTOCOL .................................................................................... 32
Related products ...................................................................................................... 36
Revision history ........................................................................................................ 37

Kit materials
The content of the kit is sufficient to perform 24 ChIP assays, 8 chromatin extractions and 8 chromatin
shearing optimization reactions. Store the components at the indicated temperature upon receipt(Table 1a).
Description

Quantity

StorageRabbit IgG

8 µl

-20°C

ChIP-seq Grade Antibody H3K4me3

8 µg

-20°C

10x Crosslinking Buffer

24 ml

4°C

Glycine

20 ml

4°C

4x Extraction buffer 1

60 ml

4°C

Extraction buffer 2

200 ml4°C

Extraction buffer 3

40 ml

4°C

10x ChIP Dilution Buffer

3.6 ml4°C

Sonication Buffer

4.8 ml4°C (Do NOT Freeze!)

DiaMag protein A-coated magnetic beads

480 µl4°C

Wash Buffer 1

4.8 ml4°C

Wash Buffer 2

4.8 µl4°C

Wash Buffer 3

4.8 µg4°C

Wash Buffer 4

4.8 µg4°C

Elution Buffer 1

4 ml

4°C (Incubate at RT before use)

Elution Buffer 2

160 µl-20°C

Carrier

64 µl

-20°C

IPure Bead v3

320 µl4°C

Wash buffer 1 w/o iso-propanol

2 ml

4°C

Wash buffer 2 w/o iso-propanol

2 ml

4°C

Buffer C

3.2 ml4°C

5 µM each/48 µl-20°C

5 µM each/48 µl

-20°C

10

RT

Arabidopsis FLC-ATG Primer pair(positive control For Arabidopsis seedlings)
Arabidopsis FLC-Intron1 Primer pair(negative control For Arabidopsis seedlings)
Crosslinking bags for plant tissue

4

UNIVERSAL PLANT CHIP-SEQ MANUALTable 1b. Components available separately
Description

Cat. No.

Quantity

Storage

Maize B73 inner stem ZmB1-UTR primer pair

C17040004

50 µl, 500 µl-20°C

Maize B73 inner stem ZmCopia primer pair

C17040005

50 µl, 500 µl-20°C

Tomato leaves SlChr2-reg8 primer pair

C17040006

50 µl, 500 µl-20°C

Tomato leaves SlChr4-NC1 primer pair

C17040007

50 µl, 500 µl-20°C

Rice seedlings OsChr4-reg9 primer pair

C17040008

50 µl, 500 µl-20°C

Rice seedlings OsMADS6 primer pair

C17040009

50 µl, 500 µl-20°C

Poplar xylem PtrMYBTF1 primer pair

C17040010

50 µl, 500 µl-20°C

Poplar xylem PtrCopia-orth primer pair

C17040011

50 µl, 500 µl

-20°C

5

Required Materials Not Provided

6•

Formaldehyde (high-quality ex: Sigmaaldrich)•

Miracloth (Calbiochem)•

Funnels (adapted to 50 ml falcon tubes)•

Vacuum pump•

Desiccator•

Liquid nitrogen

•

dH2O

•

Ceramic mortar and pestle•

50 ml falcon tubes•

1.5 ml standard tubes•

1.5 ml Safe-Lock tubes•

1.5 ml Bioruptor tubes (Depending on the Bioruptor® model used)•

200 μl strip tubes•

Bioruptor®

•

Thermomixer•

Rotating wheel•

Magnetic rack for 1.5 ml tubes•

Magnetic rack (DiaMag02 - magnetic rack (#B04000001)•

Phenol/Chloroform/Isoamylalcohol {25:24:1}•

Chloroform/Isoamylalcohol {24:1}•

ß-Mercaptoethanol•

Protease inhibitors (recommended: Protease Inhibitor Cocktailfor plant cell and tissue extracts
P 9599 from Sigma)•

Pincer, Scalpel & blades, spatula etc…

•

Towel papers

UNIVERSAL PLANT CHIP-SEQ MANUAL

Remarks before starting
The Universal Plant ChIP-seq kit is a new version of Diagenode Plant ChIP-seq kit. This version replaces
the previous one which is now discontinued. The Universal Plant ChIP-seq kit is more efficient, morereliable, easier and gives better results than the previous one. Several modifications have been madeto the protocol and the manual. Therefore, we invite you to read the current manual carefully.
Please read the complete manual very carefully before starting. It contains many details which arevery important and essential for the proper conduct of experiments. We highly recommend using thedetailed protocol for your first ChIP assays. You can use the short protocol after being experienced
with this kit.

1. Starting material

This kit has been tested and validated for both monocots and dicots plants. It was optimized for manymodel plant species belonging to many plant families and several types of tissues (see table 2).
Depending on the plant tissue and/or species, the sampling method can be different. In the majorityof cases, we recommend cutting samples into small pieces in order to facilitate the fixation withformaldehyde. A cold support such as a glass plate on ice can be used to cut the samples:
Arabidopsis seedlings (two weeks old) can be crosslinked intact. There is no need to cut them before
crosslinking. Arabidopsis adult rosette leaves can be sampled from 25 days old seedlings (18 leaves
stage) and cut to small pieces of approximately 5 mm.
For maize cut the inner stem of 2-3 weeks old seedlings into “cylinders” of approximately 2 to 3 mm
thick using a sterile blade. For maize husk material, about 3 months old plants are used. The husks
are sampled when the first silks begin to emerge from female inflorescence. The outer lignified layers
are discarded. The rest of the hush is cut into pieces of about 2 to 3 mm thick using a sterile blade.
For rice cut the seedlings into fragments of about 3 to 4 mm using a blade or sterile scissors thenhomogenise all organs (leaves, roots …) with a pincer.
For tomato, cut young leaves into “discs” of about 4 to 5 mm using a blade or sterile scissors.
Tomato anthesis are sampled during the meiotic phase (buds between 3 to 6 mm). There is no needto cut anthesis.
For in vitro poplars, cut the aerial part of the plant (leaves and stem) into pieces of about 2 to 4 mm
and homogenize pieces from different organs.
For poplar plants grown in greenhouse, cut the stem at 1 to 2 cm above the culture substrate using
loppers. Remove the apical part of the plant which has a thin and soft stem and keep the rest. Remove
the leaves and make a longitudinal “wound” along the stem using a sterile blade, this will facilitate
the removal of the bark. Before removing the bark, cut the stem into “cylinders” of about 10 cm using
loppers and keep them on ice. The bark can be removed immediately before the crosslinking.
For poplars grown in field, cut young branches that have a stem diameter greater than 1 cm using
loppers. Prepare samples exactly as described for poplar plants grown in greenhouse. If you work on
the same or similar plant material, the sampling method can be similar to this description. If you workon a completely different tissue, the sampling procedure should be optimized.
7

The optimal weight of plant tissue for ChIP experiments depends on many factors:
•
•
•

•
•

Plant species and/or families: ex herbaceous vs woody plants.
Plant organs: different organs (leaves, roots, flowers etc…) may contain different cell
types that yield more/less chromatin.
Age and/or size of the cells: the younger cells are usually smaller with a thinner cell wall,
therefore, for the same weight of plant material, the younger tissue contains more cells
and by consequence more nuclei/chromatin that are more easily available compared toolder cells.
Type of the cells: ex somatic cells vs sex organs.
The genome size: plants with a bigger genome will yield more chromatin with fewer
number of cells.

This protocol has been optimized for chromatin extraction from 0.1 to 2 g fresh weight (fw)of different plant materials (see examples in table 2). It is possible to start with more than 2g of plant material; however, the volumes of extraction and sonication buffers should be
optimised. One ChIP reaction is performed using 40 µl of the extracted chromatin.
Consequently, many ChIP reactions can be processed from 1 chromatin extraction
depending on the final volume of chromatin.
Table 2. Optimal conditions for ChIP experiments depending on the plant tissues
Plant material
Arabidopsis seedlings
Arabidopsis thaliana ecotype columbia (col-0)
Arabidopsis adult rosette leaves
Arabidopsis thaliana ecotype columbia (col-0)
Tomato leaves
Solanum lycopersicum cv. Micro-Tom
Tomato anthesis
Solanum lycopersicum
Maize inner stem
Zea mays cv. B73
Maize husk ****
Zea mays cv. B73
Rice seedlings (2-3 leaves stage)
Oriza sativa ssp Japonica cv. Nipponbare
In vitro poplar plants (leaves + stem)
Populus trichocarpa clone Nisqually
Populus tremula x alba clone INRA 717-1B4
Poplar (stem-differentiating xylem)
Populus trichocarpa clone Nisqually
Populus tremula x alba clone INRA 717-1B4OptimalCrosslinking

Sonication

Centrifuge

weight*duration(cycles)**

speed***

1g

15 min81000 x g

1g

15 min81000 x g

1g

10 min6-82100 x g

65 mg

10 min6-84000 x g

1.5 g

10 min8-102900 x g

1.5 g

10 min8-102900 x g

2g

15 min8-102900 x g> 0.1 g

15 min82900 x g

2g

15 min102900 x g* The weight indicated in the table is optimized for 1 chromatin extraction. This weight was tested and validated in ChIP using this kit.
** The number of cycles (1 cycle = 30 s ON / 30 s OFF) indicated in the table is valid only when a Bioruptor® Pico is used for sonication(see Remarks before starting, section 3)*** The indicated values correspond to the centrifuge speed during the chromatin extraction step after adding the Extraction buffer 1, 2
and 3.
**** For all plant materials cited in table 2, the concentration of formaldehyde in the crosslinking buffer is 1%, except for maize husk
material for which the concentration of formaldehyde is 3 %.

8

UNIVERSAL PLANT CHIP-SEQ MANUAL

2. Crosslinking of plant tissue

Crosslinking is one of the major steps in ChIP experiments. It allows the fixation of proteins (histones,
transcription factors etc…) to DNA by the fixation agent (ex: formaldehyde). Ideally, crosslinking is
performed in vivo using fresh tissue in order to obtain the most accurate biological information
relative to the research questions. This kit was therefore optimized on fresh/living plant material. We
note that it may be possible to crosslink frozen samples. For this, the crosslinking/sampling conditions
have to be optimized.
Inefficient crosslinking causes DNA loss and/or reduced antigen availability in chromatin.
However Over-crosslinking can result in inefficient shearing and in non-specific binding
events. The optimal cross-linking time for maximal specificity and efficiency of ChIP should
be determined empirically. A crosslinking time course (ex: 5-10-15 min) can be performed.
For the majority of plant tissues, crosslinking is performed under vacuum which permits amore efficient fixation. In fact, the cell wall may inhibit the penetration of formaldehyde.
The Crosslinking buffer provided in this kit was optimized to yield more chromatin after
nuclei preparation in combination with the chromatin extraction protocol of this kit.
Crosslinking of plant material is a delicate step, especially the washes following the fixation(see STEP 1. Crosslinking of plant tissue, 7). When removing the remaining crosslinking
buffer after each wash, it is very difficult to avoid losing plant material as small tissue
fragments can be lost. To render this step easy to perform, we provide in the kit crosslinking
bags for plant tissue with an easy closure system. Crosslinking bags will avoid any loss ofmaterial and will significantly facilitate the crosslinking, the washes and sample handlingafter fixation. Please note that provided crosslinking bags are not adapted to powder (groundplant material).

3. Shearing optimization and sheared chromatin analysis

Before starting the ChIP, the chromatin should be sheared to fragments ranging between 200 and
600 bp approximately (see figure 1 below). The shearing conditions mentioned in the protocol were
optimized using the Bioruptor® Pico (#B01060001) in combination with a water cooling system(#B02010003; 115V or #B02010002; 230V). We recommend using 1.5 ml Bioruptor® Microtubes with
Caps (#C30010016) for use with the Bioruptor® Pico. If you use a different sonicator, the sonication
conditions must be optimized accordingly. The shearing conditions mentioned in the protocol are
adequate for chromatin extracted from plant tissues we tested using this kit. If you work with adifferent plant tissue(s) the sonication conditions should be optimized for each tissue systematically
before processing a large number of samples. If you work with similar plant material, these conditions
will help you to approach the optimal sonication conditions for your tissue. However, we highly
recommend optimizing the shearing conditions before doing any new experiment. In fact, theshearing efficiency depends on numerous factors other than the sonication device and the plantmaterial such as the sample volume, the chromatin concentration and/or density, the fixation time,
etc…
An initial time-course experiment of 6-8-10-12 sonication cycles 30’’ ON/30’’ OFF is recommended
when starting a new ChIP project using the Bioruptor Pico. In some experimental conditions, even a
shorter sonication time of 2-3 cycles might result in satisfactory chromatin shearing. As best practice,choose the shortest sonication time resulting in a satisfactory ChIP efficiency (highest
recovery/lowest background). Avoid over-sonication, as it may lead to a drop of efficiency in ChIP
9

experiments, especially when non-histone proteins are to be evaluated by ChIP. A guide for successful
chromatin preparation using the Bioruptor® Pico is available on the Diagenode website.
A protocol to assess the shearing efficiency can be found in the “Additional Protocols” section. To
identify the optimal shearing conditions before ChIP experiments we offer a shearing optimization
kit (C01020014).
Figure 1. The figure shows gel electrephoresis of sheared chromatin of 3
independent nuclei preparations from maize inner stem. The sonication
was performed using Bioruptor® Pico (8 cycles: 30s ON / 30s OFF).

4. Magnetic beads

This kit includes DiaMag protein A-coated magnetic beads (cat. No. C03010020). Make sure the beads
do not dry during the procedure as this will result in reduced performance. Keep the beads in a
homogenous suspension at all times when pipetting. Variation in the amount of beads will lead tolower reproducibility. Do not freeze the beads. The amount of beads needed per IP depends on theamount of antibody used for the IP. The protocol below uses 20 μl of beads. The binding capacity ofthis amount is approximately 5 μg of antibody. With most of Diagenode’s high quality ChIP-seq grade
antibodies the recommended amount to use is 1 to 2 μg per IP reaction. However, if you plan to use
more than 5 μg of antibody per IP we recommend increasing the amount of beads accordingly.

5. DNA purification after ChIP

The DNA purification after ChIP reaction is performed using the IPure kit v2 (#C03010015) includedin this kit in this protocol (STEP4. Elution, de-crosslinking and DNA isolation). The IPure kit v2 gives
high quality DNA for PCR and library preparation for next generation sequencing. It was also tested
and validated for all plant tissues / or species indicated in table 2.
It’s highly recommended to quantify the eluted DNA using a Qubit for example. This will give a firstidea about the success of ChIP as the recovered DNA quantity should be different between IgG or no
antibody control (low), Input (high) and sample of interest (usually in between).

6. Quantitative PCR analysis and data interpretation

For qPCR analysis, depending on the concentration/quantity of immunoprecipitated DNA, use 3 μl of
purified DNA and Input samples as template. The quantity of immunoprecipitated DNA for one PCR
reaction is generally comprised between 100 and 500 pg (indicative values). However it still possible
to perform PCR using 30 pg or up to 3 ng of DNA in some cases. You can follow the manufacturer’s
recommendations relative to template DNA concentration depending on your qPCR system and/or
reagents.
10

UNIVERSAL PLANT CHIP-SEQ MANUALThe efficiency of chromatin immunoprecipitation of particular genomic loci can be expressed as therecovery of that locus calculated as the percentage of the input (% Input: the relative amount of
immunoprecipitated DNA compared to input DNA). The enrichment of immunoprecipitated DNA on
target fragments can also be calculated in comparison with the negative control (IgG or No Antibody
control). In this case, it’s considered as Fold Enrichment. The enrichment can also be normalized to areference gene (Normalized Enrichment). See below formulas for each type of calculation. Theseequations assume that the PCR is 100 % efficient (amplification efficiency = 2). For accurate results
the real amplification efficiency, if known, should be used.
NB: It is important to verify the Melting Peaks of each primer for each sample before any calculation.
For some samples, especially IgG negative control, when DNA quantity is low, the primers may havetendency to make dimers resulting in a false peak (and as a consequence a false Ct value). In order
to avoid this problem, use enough DNA for PCR. The Melting Peaks of IgG and samples should besuperposed with those of the Input (which generally has a higher DNA concentration).
% Input = (100/Df) x [2^(Ct Input - Ct Sample)]
Df: dilution factor of the Input i.e. if Input is 1% (see STEP 3. Magnetic Immunoprecipitation, 33) the Df is 100.
If Input is 10 %, the Df is 10 etc…
Ct: Ct values are the threshold cycles from the exponential phase of the qPCR
Fold Enrichment = 2^(Ct Sample – Ct Nc)
Nc: negative control IgG or No Antibody control
Normalized Enrichment = 2^(Ct Sample – Ct Reference gene)

7. Negative and positive IP controls

The kit contains a negative (IgG, cat. No. C15410206) and a positive (H3K4me3, cat. No. C15410003)
control antibody. We recommend including one IgG negative IP control in each series of ChIP
reactions. We also recommend using the positive control ChIP-seq grade H3K4me3 antibody at least
once. The kit contains qPCR primer pairs for amplification of a positive and negative control target
for H3K4me3 in Arabidopsis thaliana (Arabidopsis FLC-ATG and Arabidopsis FLC-Intron1,
respectively). Positive and negative control primer pairs for H3K4me3 in other plant species are alsoavailable but not included in the kit. They can be purchased separately (see table 1b).

8. Preparation of buffers

Check Elution buffer 1 for precipitation upon storage. If necessary, warm to 37°C until the precipitate
has fully dissolved.
Crosslinking buffer and ChIP Dilution Buffer are provided as 10x concentrates. Extraction buffer 1 isprovided as 4x concentrate. Before use prepare the necessary amount of 1x buffer using dH20.
Protease inhibitors, PMSF, and β-mercaptoethanol should be added freshly, where stated.

11

SHORT PROTOCOLSTEP 1

Crosslinking of plant tissue1.

Prepare 1x Crosslinking Buffer containing 1 % formaldehyde and store it on ice.
1x Crosslinking buffer
10x Crosslinking buffer 3 ml

12

Formaldehyde

1 % (ex: 811 μl of Formaldehyde solution 37 %)dH2o

Up to 30 ml

Total volume

30 ml

2.

Harvest the plant material and wash it with dH2O. Dry it using towel papers and keep it on ice.

3.

Weigh the plant material and put it in a crosslinking bag. Close and keep on ice.

4.

Introduce the crosslinking bag to the crosslinking buffer in 50 ml Falcon tube.

5.

Crosslink under vacuum in a desiccator pre-filled with ice during 10 to 15 minutes.

6.

Replace 2.5 ml of the crosslinking buffer by 2.5 ml of Glycine. Mix the solution and apply
vacuum for additional 5 minutes.

7.

Discard the Crosslinking Buffer and wash 3 times with cold deionized water.

8.

Dry plant material using paper towels and put it into a new 50 ml tube. Close the tube andsnap-freeze in liquid nitrogen.

UNIVERSAL PLANT CHIP-SEQ MANUALSTEP 2Chromatin extraction from plant tissue
and chromatin shearing
9.

Calculate the necessary amount of 4x Extraction buffer 1 (7.5 ml/sample), 1x Extraction buffer
2 (25 ml/sample) and Extraction buffer 3 (5 ml/sample).

10. Prepare complete 1x Extraction buffer 1 by diluting the 4x Extraction buffer 1, 1x Extraction
buffer 2 and Extraction buffer 3 as indicated below and keep them on ice.
Complete Extraction buffer 1(Volume for 1 sample)

4x Extraction buffer 1

7.5 mldh2oProtease inhibitors
ß-Mercaptoethanol
Phenylmethylsulfonylfluoride (PMSF)

22.5 ml
Add according to manufacturer’s instructions
Add to a final concentration of 5 mM
Add to a final concentration of 0.1 mM

Total volume

30 ml

Complete Extraction buffer 2(Volume for 1 sample)

Extraction buffer 2

25 mlProtease inhibitors

Add according to manufacturer’s instructions

ß-Mercaptoethanol

Add to a final concentration of 10 mM

Total volume

25 ml

Complete Extraction buffer 3(Volume for 1 sample)

Extraction buffer 3

5 ml

Protease inhibitors

Add according to manufacturer’s instructions

ß-Mercaptoethanol

Add to a final concentration of 10 mM

Total volume

5 ml

11. Grind the plant tissue in liquid nitrogen to a fine powder. Transfer the powder to an empty precooled 50 ml tube.
12. Take the tubes out of the liquid nitrogen, wait 15 seconds and add 30 ml of cold Complete
Extraction Buffer 1.
13. Incubate the tubes at 4°C in a rotating wheel for at least 15 min and up to 1 h.

13

14. Filter the suspension twice through 1 layer of Miracloth (Calbiochem) and collect the flowthrough in a new 50 ml tube on ice.
15. Centrifuge for 20 minutes at 4 °C at 1000 to 2900 x g (see table 2). Discard the supernatant andavoid losing any part of the pellet.
16. Wash the pellet containing the nuclei 5 times with 5 ml Complete Extraction Buffer 2. Aftereach wash, centrifuge for 5 minutes at 4 °C at the same speed used in the previous step anddiscard the supernatant.
17. Wash once with 5 ml Extraction Buffer 3 as in step 15. Centrifuge for 5 minutes at 4 °C anddiscard the supernatant.
18. Centrifuge at 4 °C for 30 seconds. Remove the rest of the extraction buffer 3 with a pipette.
19. Resuspend the pellet in 600 μl of Sonication Buffer (supplemented with protease inhibitors) by
pipetting and transfer the suspension to a 1.5 ml Safe-Lock tube.
20. Incubate the 1.5 ml tube at 4°C in a rotating wheel for 15 minutes.
21. Transfer the lysate to a sonication tube (maximum 300 μl per tube).
22. Sonicate the chromatin with the Bioruptor® Pico for 6 to10 cycles (see table 2) with the settings
30 sec ON / 30 sec OFF at 4 °C.
23. Transfer the sonicated chromatin to a standard 1.5 ml tube and centrifuge for 5 minutes at
18.000 x g at 4 °C.
24. Transfer the supernatant to a new 1.5 ml tube and check the shearing efficiency on an agarosegel. To identify correctly the fragment size distribution, sheared chromatin should be
decrosslinked (see Additional Protocols section)..

14

UNIVERSAL PLANT CHIP-SEQ MANUALSTEP 3Magnetic Immunoprecipitation
25. Identify the number of ChIP reactions (nc) you will perform (don’t consider the Input sample)
and the number of antibodies (na) you will use including IgG.
26. Prepare the necessary volume (V) of 1x ChIP Dilution buffer as indicated.
V (μl) = (nc x 302) + [600 x (na + 1)]
1x ChIP dilution buffer(maximum volume for 1 sample)

10x ChIP dilution buffer

150 μldH2o1350 μl

Protease inhibitors

Add according to manufacturer’s instructions

Total volume

1.5 ml

27. Calculate the necessary amount (ov1) of DiaMag protein A-coated magnetic beads (20 μl per
ChIP reaction) and transfer it to a 1.5 ml tube. Place the tube in the magnetic rack. Wait 1
minute and remove the supernatant by pipetting.
28. Wash the beads 3 times with 200 μl of ice-cold 1x ChIP Dilution Buffer.
29. After the last wash, resuspend the beads in the original volume + 1 μl per ChIP reaction[ov1 + (nc x 1 μl)] by adding 1x ChIP Dilution Buffer and keep the tube on ice.
30. Prepare one Safe-Lock 1.5 ml tube per antibody including negative control IgG. Add thenecessary amount (ov2) of washed beads (20 μl per ChIP reaction for each Antibody / IgG). For
each tube, if the final volume is less than 100 μl, fill up with 1x ChIP Dilution Buffer.
31. Add x μl of antibody to the beads and incubate 4h to overnight on a rotating wheel at 4°C.
32. Wash the antibody coated beads 3 times with 200 μl 1x ChIP Dilution Buffer. After the last wash
discard the supernatant and resuspend the beads in the original volume (ov2) + 1 μl per ChIP
reaction using 1x ChIP Dilution Buffer and keep the tube on ice.
33. Per ChIP reaction: Dilute 40 μl of the sheared chromatin by adding 160 μl of cold 1x ChIP
Dilution Buffer. Add protease inhibitors and mix thoroughly. Place 2 μl of the diluted chromatinaside (-20 °C) to be used as INPUT later.
34. Per ChIP reaction, add 20 μl of Antibody coated magnetic beads to the chromatin. Incubate 4h
to overnight in a rotating wheel at 4 °C.
35. After the incubation briefly spin the tubes and place them in the magnetic rack. Wait for one
minute and remove the supernatant. Wash beads once with 200 μl of wash buffer 1, 2, 3 and
4.

15

STEP 4

Elution, de-crosslinking and DNA isolation using IPure Kit v2
36. Resuspend the beads pellet with 100 μl of Elution Buffer 1 and transfer it into a clean 200 μl
strip tubes. Incubate for 30 minutes at room temperature on a rotating wheel.
37. Thaw your Input sample(s) from (step 3, 33) on ice.
38. After 30 minutes incubation, vortex, perform a short spin and put the strip on a magnetic rackand wait for 1 minute.
39. Collect the supernatant, transfer it into a new 200 μl strip tubes. Add 98 μl of Elution buffer 1
to Input sample(s) and transfer it into the 200 μl strip tubes. Add 4 μl of Elution Buffer 2 to all
samples (ChIP + Input).
40. Vortex well until the sample is clear again. Incubate the strip 4h to overnight at 65 °C.
41. Add 2 μl of carrier to each IP and input sample. Vortex briefly and perform a short spin.
42. Add 100 μl of 100% isopropanol to each IP and input sample. Vortex briefly and perform ashort spin.
43. Resuspend the provided IPure Magnetic beads and transfer 10 μl to each IP and input sample.
44. Vortex and incubate 10 minutes at room temperature on a rotating wheel (40 rpm).
45. Prepare the Wash buffer 1 and 2 by adding 2 ml of Isopropanol (100 %) for each.
Wash buffer 1
Wash buffer 1 w/o isopropanol

2 ml

Isopropanol (100%)

2 ml

Total volume

4 ml

Wash buffer 2
Wash buffer 2 w/o isopropanol

2 ml

Isopropanol (100%)

2 ml

Total volume

4 ml

46. Perform a short spin of the strip. Place the strip on a magnet and discard the supernatant.
47. Add 100 μl of wash buffer 1 to the beads pellet. Vortex well and incubate for 30 seconds on arotating wheel. Spin the strip, place it on a magnet and discard the supernatant.

16

UNIVERSAL PLANT CHIP-SEQ MANUAL

48. Perform a second wash with 100 μl of wash buffer 2. Incubate for 30 seconds on a rotating
wheel. Place on a magnet and discard the supernatant.
49. Spin the strip again and place it on the magnet. Discard any remaining wash buffer 2.
50. Resuspend the beads pellet in 25 to 100 μl of buffer C.
51. Incubate at room temperature for 15 minutes on a rotating wheel. Spin the strip and place it
on a magnet.
52. Transfer the eluate into new tubes and discard the beads. The recovered DNA can be used
immediately for PCR and/or library preparation or stored at -20 °C.

qPCR analysis and results
53. Prepare the qPCR master mix (total volume 10 μl = 7 μl PCR master mix + 3 μl DNA sample):•1 x SYBR Green PCR master mix (e.g.: iQ SYBR Green supermix)•

0.25 μl of each primer pair (5 μM) (0.5 μl of the provided primer mix)•

Fill up to 10 μl with DNase-free water

54. Use 3 μl of purified DNA (100 to 500 pg) as template for qPCR.
55. Perform the qPCR using the following conditions: 3 to 10 min denaturation step at 95°C
followed by 45 cycles of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C, followed by a Melting
Curve step: 5 s at 95 °C, 60 s at 65 °C and 1 s at 97 °C.

17

18

STEP 1 – Crosslinking of plant tissue

19

STEP 2 – Chromatin extraction from plant tissue and chromatin shearing

21

STEP 3 – Magnetic Immunoprecipitation

24

STEP 4 – Elution, de-crosslinking and DNA isolation using IPure Kit v2

26

STEP 5 -qPCR analysis and results

28

UNIVERSAL PLANT CHIP-SEQ MANUALSTEP 1

Crosslinking of plant tissue
CAUTION: Steps 1, 4, 5, 6 and 7 should be carried out under the fume hood!
1. Prepare 1x Crosslinking Buffer containing 1 % formaldehyde and store it on ice.
1x Crosslinking buffer
10x Crosslinking buffer

3 mlFormaldehyde

1 % (ex: 811 μl of Formaldehyde solution 37 %)dH2o

Up to 30 ml

Total volume

30 ml

2. Harvest the plant material (a description of the sampling procedure of each tested tissue and/or
species is available in the section remarks before starting, Starting material). Wash the plantmaterial with dH2O after harvesting. Dry it using towel papers and keep it on ice until the
crosslinking step in order to protect tissues from dryness. Plant material should not be in directcontact with ice.
3. Weigh the necessary quantity of plant material mentioned in table 2 and starting material
section. If the goal is to optimise the shearing efficiency or to compare many treatment
conditions the weight should be exactly the same for all samples in order to have reliable results.
Put the weighted sample in a crosslinking bag, make a node and pull simultaneously both endsof the thread. A second node can be made in order to close correctly the bag. Keep the
crosslinking bag with the sample inside on ice until starting the crosslinking.
4. Introduce the closed crosslinking bag to the 50 ml falcon tube containing the crosslinking
solution supplemented with formaldehyde. Make sure that the bag is submerged in buffer and
not floating on top of it. A clean stainless steel paper clip can be fixed in the top of thecrosslinking bag. This will somehow “lock” the crosslinking bag in the bottom of the tube andprevent it from floating on top of the solution. All samples should be introduced simultaneously
to the 50 ml falcon tubes containing the crosslinking buffer supplemented with formaldehyde.
Prolonged incubation of samples weighted first in crosslinking solution before applying thevacuum may induce variability.
5. Put the 50 ml falcon tubes containing samples in the desiccator (pre-filled with ice). During thecrosslinking under vacuum, the tubes have to be opened. Otherwise, vacuum cannot be applied
to the plant material. Close the desiccator lid and crosslink the plant tissue under vacuum (~ 950 Millibars) during 10 to 15 min depending on plant tissue and/or specie (see table 2). If you
use a different plant material and/or a different vacuum pressure, the crosslinking conditions
should be optimized before doing any large scale fixation.

19

6. Turn off the vacuum pump and release the vacuum slowly. Replace 2.5 ml of the crosslinking
buffer by 2.5 ml of Glycine. Mix the solution and apply vacuum for additional 5 min. Release thevacuum slowly.
7. Discard the Crosslinking Buffer following the legislation relative to dangerous chemical productsof your country. Add 40 ml of cold deionized water (4 °C; not supplied), close the tube and mix.
Discard the water with dangerous products as it may contain formaldehyde. Repeat the washing
step twice (3 washes in total).
8. Take the crosslinking bag out of the 50 ml falcon tube. Open the bag or cut it with scissors,
remove plant tissue and dry it thoroughly using paper towels. Insert the plant tissue into new
50 ml tubes, close the tubes and snapfreeze them in liquid nitrogen. At this point the tissue canbe stored at - 80 °C for several months or directly used for chromatin extraction.
NB: For poplar plants grown in greenhouse and/or field, remove the bark from stem fragments. Placea clean box (ex: clean empty tips box) on ice in the desiccator and fill it with stem fragments. Add thenecessary volume (v1 ml) of 1x Crosslinking Buffer supplemented with formaldehyde until all
fragments are covered by the solution. A clean weight can be placed on top of the box to prevent
stem fragments from floating. Crosslink the poplar tissue under vacuum (+/- 950 Millibars) during 15
min. If you use a different vacuum pressure, the crosslinking time should be optimized.
Turn off the vacuum pump and release the vacuum slowly. Remove a volume (v2) (v2 = 0.08 * v1 ml)
from the crosslinking buffer and replace it with Glycine solution (ex: from 100 ml crosslinking solution
remove 8 ml and add 8 ml of Glycine) to stop the crosslinking. Apply vacuum for additional 5 min.
Release the vacuum slowly.
Discard the Crosslinking Buffer and wash the stem fragments 3 times generously with cold deionizedwater. Place the fragments on towel paper.
Scrape gently the outermost layer of stem fragments corresponding to the stem-differentiating xylem(SDX) using a blade. The scraped SDX should be collected in a clean recipient containing liquid
Nitrogen until the end of the sampling. Weigh the SDX and store in aliquots of 2 g each at -80°C up
to several months.

20

UNIVERSAL PLANT CHIP-SEQ MANUALSTEP 2Chromatin extraction from plant tissue
and chromatin shearing
CAUTION: Buffers containing ß-mercaptoethanol should be prepared, handled and discarded under
the fume hood!

9. Calculate the necessary amount of Extraction buffer 1 (7.5 ml/sample), Extraction buffer 2 (25ml/sample) and Extraction buffer 3 (5 ml/sample).
10. Prepare complete 1x Extraction buffer 1 by diluting the 4x Extraction buffer 1, Extraction
buffer 2 and Extraction buffer 3 containing protease inhibitors, ß-mercaptoethanol and PMSF
(only for Extraction buffer 1) as indicated below and keep them on ice.
Complete Extraction buffer 1(Volume for 1 sample)

4x Extraction buffer 1

7.5 mldh2oProtease inhibitors
ß-Mercaptoethanol
Phenylmethylsulfonylfluoride (PMSF)

22.5 ml
Add according to manufacturer’s instructions
Add to a final concentration of 5 mM
Add to a final concentration of 0.1 mM

Total volume

30 ml

Complete Extraction buffer 2(Volume for 1 sample)

Extraction buffer 2

25 mlProtease inhibitors

Add according to manufacturer’s instructions

ß-Mercaptoethanol

Add to a final concentration of 10 mM

Total volume

25 ml

Complete Extraction buffer 3(Volume for 1 sample)

Extraction buffer 3

5 ml

Protease inhibitors

Add according to manufacturer’s instructions

ß-Mercaptoethanol

Add to a final concentration of 10 mM

Total volume

5 ml

11. Fill a ceramic mortar with liquid nitrogen and add the plant tissue. Grind the plant tissue
thoroughly using a mortar and a pestle to a fine powder in liquid nitrogen. Avoid letting thetissue to thaw at any time. Transfer the powder to an empty pre-cooled 50 ml tube. As it is21

difficult to transfer all powder using pre-cooled spatula, you can use a small volume of liquid
Nitrogen to transfer the rest of the powder. Make sure that all the liquid Nitrogen is evaporated
completely before closing the tube. Close the tube and keep it in liquid Nitrogen. Repeat thisprocedure for the other samples.
12. When all samples are grinded, take the tubes out of the liquid nitrogen, wait 15 seconds andadd 30 ml (20 ml if powder weight is lower than 0.5 g) of cold Complete Extraction Buffer 1. It
is important to add the Extraction buffer 1 simultaneously to all samples to prevent variability.
Vortex vigorously until the tissue is completely homogenized in the buffer.
13. Incubate the tubes at 4°C in a rotating wheel for at least 15 minutes and up to 1 h. This will
improve the efficiency of the chromatin extraction. The Extraction buffer 1 will lysate the cells
but not the nuclear membrane and will permit a better separation between cell lysates andnuclei.
14. Filter the suspension twice through 1 layer of Miracloth (Calbiochem) and collect the flowthrough in a new 50 ml tube on ice. The pore size of Miracloth layer is about 22 μm. The nuclei
having around 10 μm diameter will pass through the pores but not the bigger cell lysates.
15. Centrifuge for 20 minutes at 4 °C at 1000 to 2900 x g depending on plant material (see table 2).
The nuclei being heavier than the remaining cell lysates will remain in the bottom of the falcon
tube after centrifugation. Discard the supernatant and avoid losing any part of the pellet.
16. Wash the pellet containing the nuclei 5 times with 5 ml Complete Extraction Buffer 2. In eachwashing step carefully resuspend the pellet by gentle shaking (no vortexing or pipetting). The
pellet may be difficult to resuspend. Thus alternate short shaking with incubation on ice for 15
seconds. This will help to resuspend the nuclei. If the problem persists, you can use a tip for
example to mechanically scatter the pellet. After each wash, centrifuge for 5 minutes at 4 °C at
the same speed used in the previous step and discard the supernatant without disturbing thepellet.
17. Wash once with 5 ml Extraction Buffer 3 as in step 15. Centrifuge for 5 minutes at 4 °C anddiscard the supernatant.
18. Centrifuge at 4 °C for 30 seconds. Remove very carefully the rest of the extraction buffer 3 witha pipette. Remove only the clear liquid without disturbing the pellet. If the liquid is not clear you
can centrifuge again for 30 seconds. It is important to remove all the remaining liquid before
adding the sonication buffer. Keep the tubes with the pellets on ice. At this step, the nuclei pellet
can be stored at - 80 °C.
19. Resuspend the crude nuclear pellet in 600 μl of Sonication Buffer (supplemented with proteaseinhibitors) by pipetting and transfer the suspension to a 1.5 ml Safe-Lock tube. If you started
with less than 0.5 g of plant material you can resuspend the pellet in 300 μl. This volume will
determine the chromatin concentration and will, by consequense, affect the shearing efficiency.
It is an important factor to be taken into consideration when optimizing the shearing conditions.
20. Incubate the 1.5 ml tube at 4°C in a rotating wheel for 15 minutes. During this step, thesonication buffer will permit the lysis of the nuclear membrane before sonication making thechromatin more accesible for sonication.

22

UNIVERSAL PLANT CHIP-SEQ MANUAL

21. Transfer the lysate to a sonication tube (tubes depend on the Bioruptor® model used). Be
careful not to cause bubbles by pipetting. Add maximum 300 μl per tube. If the volume is bigger
than 300 μl, split it in as many sonication tubes as necessary.
22. Sonicate the chromatin with the Bioruptor® Pico for 6 to10 cycles depending on the plant tissue(see table 2) with the settings 30 sec ON / 30 sec OFF at 4 °C. Optimization may be required
depending on the tissue type, fixation time and Bioruptor® model used. We recommend
performing a time course experiment in order to specify the optimal sonication conditions (seeRemarks before starting, 3).
23. Transfer the sonicated chromatin to a standard 1.5 ml tube and centrifuge for 5 minutes at
18.000 x g at 4 °C.
24. Transfer the supernatant to a new 1.5 ml tube. We recommend highly measuring theconcentration of chromatin after sonication using a Qubit (DNA). Following the above
procedure, you should obtain between 2 and 30 μg (indicative values). This will permit toidentify the chromatin yield and to estimate the number of cells (N) corresponding to theobtained chromatin. Therefore, the number of cells that will be used for each ChIP experiment(n) can be calculated (see formulas below).
(N) = (0.978 x Q x V x 10^6) / Gs
A calculation table is available for downloading in Diagenode website (same location as the
manual).
(n) = (N/V) x 40
• N: number of cells for 1 chromatin extraction• 0.978: conversion factor from bp to pg (1 pg = 0,978 * 10^9 bp)• Q: concentration of sonicated chromatin (ng/μl)• V: volume of sonication buffer used to resuspend nuclei pellet• Gs: Genome size (Mbp)• n: number of cells for 1 ChIP reaction• 40: volume of chromatin for 1 ChIP reaction
Purify the DNA from 50 μl of chromatin to check the shearing efficiency on an agarose gel. To
identify correctly the fragment size distribution, sheared chromatin should be decrosslinked(see Additional Protocols section).
At this point the chromatin can be stored at - 80 °C or directly used for the immunoprecipitation.
Avoid multiple freeze/thaw cycles.
NB: Not sheared chromatin from step 20 can also be stored at -80°C and sheared after thawing

23

STEP 3Magnetic Immunoprecipitation
The chromatin extraction from plant material using this kit yields up to 600 μl of sheared chromatin.
This protocol has been optimized for 40 μl of chromatin per ChIP reaction. Although it is possible touse more chromatin, we recommend performing separate IP reactions and pooling of the samples
prior to DNA purification. Please note that we recommend including one negative control in eachexperiment (ChIP with the IgG negative control).
25. Identify the number of ChIP reactions (nc) you will perform. At this step, don’t consider the Input
sample.Identify the number of antibodies (na) you will use in your experiments including IgG orno antibody control.
26. Prepare the necessary volume (V) of 1x ChIP Dilution buffer by diluting the 10x ChIP Dilution
buffer and supplement it with protease inhibitors. You will need maximum 1.5 ml of 1x ChIP
Dilution Buffer for 1 ChIP reaction.
1x ChIP dilution buffer(maximum volume for 1 sample)

10x ChIP dilution buffer

150 μldH2o1350 μl

Protease inhibitors

Add according to manufacturer’s instructions

Total volume

1.5 mlNB: If you perform many ChIP reactions at the same time, you will need less volume per ChIP.
You can use the following formula to calculate exactly the volume of 1x ChIP dilution buffer
you will need:
V (μl) = (nc x 302) + [600 x (na + 1)]
27. Calculate the necessary amount (this will be the original volume (ov1 - see 29) of DiaMag protein
A-coated magnetic beads (20 μl per ChIP reaction) and transfer it to a standard 1.5 ml tube.
Place the tube in the magnetic rack. Wait 1 minute to allow the beads to be captured by themagnet and remove the supernatant by pipetting.
28. Wash the beads 3 times with 200 μl of ice-cold 1x ChIP Dilution Buffer. For each wash, add 200
μl of 1x ChIP Dilution Buffer, resuspend by pipetting up and down several times and place thetubes in the magnetic rack, wait 1 minute or until the liquid is clear and discard the supernatant.
Alternatively, you can centrifuge the tubes for 5 minutes at 1.300 x g, discard the supernatantand keep the bead pellet.
29. After the last wash, resuspend the beads in the original volume with a margin of 1 μl per ChIP
reaction [ov1 + (nc x 1 μl)] by adding 1x ChIP Dilution Buffer and keep the tube on ice.

24

UNIVERSAL PLANT CHIP-SEQ MANUAL

30. Prepare one Safe-Lock 1.5 ml tube per antibody including negative control IgG. At this step,
there is no need to use one tube for each ChIP reaction. Add the necessary amount (ov2) of
washed beads (20 μl per ChIP reaction for each Antibody / IgG). Always resuspend beads before
transferring to tubes. For each tube, if the final volume is less than 100 μl, fill up the volume
with 1x ChIP Dilution Buffer.
31. Add x μl of antibody to the beads and incubate on a rotating wheel overnight at 4°C (or at least
4 hours). The quantity of antibody depends on its quality. The quantity of IgG should be similar
to that of the antibody. If you use the anti-H3K4me3 antibody provided in the kit, use 1 μg
antibody per ChIP reaction and the same amount of IgG for the negative control.
32. Wash the antibody coated beads (as in point 27) 3 times with 200 μl 1x ChIP Dilution Buffer.
After the last wash discard the supernatant and resuspend the beads in the original volume (ov2
- see 30) with a margin of 1 μl per ChIP reaction using 1x ChIP Dilution Buffer and keep the tube
on ice.
33. Per ChIP reaction: Dilute 40 μl of the sheared chromatin in a ratio of 1:5 by adding 160 μl of cold
1x ChIP Dilution Buffer. Add protease inhibitors and mix thoroughly. Scale according to thenumber of ChIP reactions performed with the same chromatin (make a mix). Place 2 μl (1 % of
the volume of chromatin for 1 ChIP) of the diluted chromatin aside (-20 °C) to be used as INPUT
later.
34. Prepare as many Safe-Lock 1.5 ml tubes as the number of ChIP reactions. Add 200 μl of diluted
chromatin and 20 μl of Antibody coated magnetic beads. Incubate overnight (or at least 4h) ina rotating wheel at 4 °C.
35. After the incubation briefly spin the tubes and place them in the magnetic rack. Wait for one
minute and remove the supernatant. Wash beads once with 200 μl of wash buffer 1, 2, 3 and 4.
For each wash: add 200 μl of the washing buffer, resuspend the beads by inverting the tubes
gently (do not resuspend by pipetting), incubate 5 minutes at 4°C in a rotating wheel, short spin,
place in the magnetic rack, wait 1 minute and discard the supernatant.

25

STEP 4

Elution, de-crosslinking and DNA isolation
using IPure Kit v2
NOTE: See Remarks before starting, 5: DNA purification after ChIP.
36. Add 100 μl of Elution Buffer 1 to the beads pellet. Resuspend the beads pellet and transfer it
into a clean 200 μl strip tubes. Incubate for 30 min at room temperature on a rotating wheel.
This will elute the chromatin from the DiaMag protein A-coated magnetic beads.
37. Thaw your Input sample(s) from (step 3, 33) on ice.
38. After 30 min incubation, vortex, perform a short spin and finally put the strip on a magnetic rackand wait for 1 minute.
39. Collect the supernatant, transfer it into a new 200 μl strip tubes. Add 98 μl of Elution buffer 1
to Input sample(s) and transfer it into the 200 μl strip tubes. Add 4 μl of Elution Buffer 2 to all
samples (ChIP + Input).
40. Vortex well until the sample is clear again. Incubate the strip 4h or overnight at 65 °C (in athermomixer for example) to reverse-crosslinked the chromatin.
41. Add 2 μl of carrier to each IP and input sample. Vortex briefly and perform a short spin.
42. Add 100 μl of 100% isopropanol to each IP and input sample. Vortex briefly and perform a shortspin. Following the addition of isopropanol the solution may become cloudy. This is not
detrimental to your experiment and will not influence the quality or quantity of the purified
DNA.
43. Resuspend the provided IPure Magnetic beads and transfer 10 μl to each IP and input sample.
44. Vortex and incubate 10 minutes at room temperature on a rotating wheel (40 rpm).
45. Prepare the Wash buffer 1 and 2 by adding 2 ml of Isopropanol (100 %) for each.
Wash buffer 1
Wash buffer 1 w/o isopropanol

2 ml

Isopropanol (100%)

2 ml

Total volume

4 ml

Wash buffer 2

26

Wash buffer 2 w/o isopropanol

2 ml

Isopropanol (100%)

2 ml

Total volume

4 ml

UNIVERSAL PLANT CHIP-SEQ MANUAL

46. Perform a short spin of the strip. Beads are often remaining on the side of the tube. A spin allows
the beads to be more efficiently captured. Place the strip on a magnet and discard thesupernatant.
47. Add 100 μl of wash buffer 1 to the beads pellet. Vortex well until the beads pellet is completely
broken. In order to avoid the beads pellet to be too difficult to break down, do not let the beads
for too long on the magnet. Incubate for 30 seconds on a rotating wheel. Spin the strip until all
the beads are at the bottom of the tube. Place the strip on a magnet and discard thesupernatant.
48. Perform a second wash with 100 μl of wash buffer 2. Incubate for 30 seconds on a rotating
wheel. Place on a magnet and discard the supernatant.
49. Spin the strip again and place it on the magnet. Discard any remaining wash buffer 2.
50. Resuspend the beads pellet in 25 to 100 μl (depending on plant material and expected DNA
concentration after IP) of buffer C. If you expect low amount of DNA, use 25 μl of buffer C.
51. Incubate at room temperature for 15 minutes on a rotating wheel. Spin the strip and place it ona magnet.
52. Transfer the eluate into new tubes and discard the beads. The recovered DNA can be used
immediately for PCR and/or library preparation or stored at -20 °C.

27

STEP 5qPCR analysis and results
We recommend analysing the Immunoprecipitated DNA by qPCR using at least 1 positive and 1
negative control target.
53. Prepare the qPCR master mix (total volume 10 μl = 7 μl PCR master mix + 3 μl DNA sample):• 1 x SYBR Green PCR master mix (e.g.: iQ SYBR Green supermix)• 0.25 μl of each primer pair (5 μM) (0.5 μl of the provided primer mix)
• Fill up to 10 μl with DNase-free water
54. Use 3 μl of purified DNA (100 to 500 pg) as template for qPCR. For more details refer to thesection: Remarks before starting, Quantitative PCR analysis and data interpretation.
55. Perform the qPCR using the following conditions: 3 to 10 min denaturation step at 95°C (pleasecheck carefully the supplier’s recommendations concerning Taq polymerase activation time),followed by 45 cycles of 30 s at 95°C, 30 s at 60 °C (temperature depending on primers) and 30
s at 72 °C, followed by a Melting Curve step: 5 s at 95 °C, 60 s at 65 °C and 1 s at 97 °C. These
conditions may require optimisation depending on the type of Master Mix or qPCR system used.
For qPCR results and data interpretation, refer to the section: Remarks before starting,
Quantitative PCR analysis and data interpretation.

Figure 2. ChIP was performed on
Arabidopsis thaliana (Col-0) seedlings 14
days after germination using our premiumanti-H3K4me3 ChIP-seq grade antibody(Cat. No. C15410003) and rabbit IgG (cat.
No. C15410206). Error bars represent thestandard deviation of at least 3 biologicalreplicates. 1 µg of H3K4me3 antibody and1 µg of the negative IgG control were used
per ChIP reaction. Quantitative PCR was
performed with the positive control FLCATG and the negative control FLC-Intron1
primer sets from the kit. The recovery isexpressed as % of input.

28

UNIVERSAL PLANT CHIP-SEQ MANUAL

Figure 3. ChIP was performed on Zea mays
cv, B73 inner stem obtained from 3 leaves
stage seedlings using our premium antiH3K4me3 ChIP-seq grade antibody (cat.
No. C15410003) and rabbit IgG (cat. No.
C15410206). Error bars represent thestandard deviation of at least 3 biologicalrepetitions. 1 µg of H3K4me3 antibody
and 1 µg of the negative IgG control wereused per ChIP reaction. Quantitative PCR
was performed with the positive control
b1-UTR and the negative control Copia
primer sets available separately. The
recovery is expressed as % of input.

Figure 4. ChIP was performed on Oriza
sativa ssp Japonica cv. Nipponbare 3
leaves stage seedlings using our premiumanti-H3K4me3 ChIP-seq grade antibody(cat. No. C15410003) and rabbit IgG (cat.
No. C15410206). Error bars represent thestandard deviation of at least 3 biologicalrepetitions. 1 µg of H3K4me3 antibody
and 1 µg of the negative IgG control wereused per ChIP reaction. Quantitative PCR
was performed with the positive control
Cent4-reg9 and the negative control
MADS6 primer sets available separately.
The recovery is expressed as % of input

Figure 5. ChIP was performed on
Solanum lycopersicum cv Micro-Tom
young leaves using our premium antiH3K4me3 ChIP-seq grade antibody(#C15410003)
andrabbitIgG(#C15410206). Error bars represent thestandard deviation of at least 3 biologicalrepetitions. 1 µg of H3K4me3 antibody
and 1 µg of the negative IgG control wereused per ChIP reaction. Quantitative PCR
was performed with the positive control
Chr2-reg8 and the negative control Chr4NC1 primer sets available separately. The
recovery is expressed as % of input.
29

Figure 6. ChIP was performed on young xylem ofPopulus trichocarpa clone Nisqually using our
H3K4me3 antibody (#C15410003) and rabbit IgG(#C15410206). Error bars represent the standard
deviation of at least 3 biological repetitions. 1 µgof H3K36me3 antibody and 1 µg of the negative
IgG control were used per ChIP reaction.
Quantitative PCR was performed with the positive
control Ptr-MYBTF1 and the negative control PtrCopia-orth primer sets available separately. The
recovery is expressed as % of input.

30

UNIVERSAL PLANT CHIP-SEQ MANUAL

31

32

UNIVERSAL PLANT CHIP-SEQ MANUALSheared chromatin analysis
Reagents not supplied:•
RNase cocktail (e.g. Ambion, AM 2286 A)•
Phenol/chloroform/isoamyl alcohol {25:24:1}•
Chloroform/isoamyl alcohol {24:1}•
100 % Ethanol•
70 % Ethanol•
DNA precipitant (Cat. No. C03030002)•
DNA co-precipitant (Cat. No. C03030001)•
Agarose and TAE buffer•
DNA ladder marker•
DNA loading dye

CAUTION: Steps 8-12 should be carried out under the fume hood!
1.

Use 50 µl aliquot of sheared chromatin from Step 2 point 24 to analyse the shearing
efficiency.

2.

Prepare RNase cocktail dilution (e.g. Ambion, AM 2286 A: dilute 1µl of cocktail in 150 µl of
ChIP-seq grade water).

3.

Add 2 µl of diluted RNase cocktail.

4.

Incubate 1h at 37°C.

5.

Add 50 µl of elution buffer 1.

6.

Add 4 µl of elution buffer 2, mix thoroughly.

7.

Incubate samples at 65°C for 4h (or overnight).

8.

Extract DNA once with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1).
Incubate the sample at RT for 10 min on a rotating wheel.

9.

Centrifuge for 2 min at 13,000 rpm at RT. Transfer the top aqueous phase into a new 1.5 ml
tube.

10. Add 1 volume of chloroform/isoamyl alcohol (24:1). Incubate the sample at RT for 10 min on
a rotating wheel.
11. Centrifuge for 2 min at 13,000 rpm at RT. Transfer the top aqueous phase into a new 1.5 ml
tube.
12. Precipitate the DNA by adding 10 µl DNA precipitant, 5 µl of co-precipitant, and 500 µl of
cold 100% ethanol to the sample. Incubate at -80 °C for 30 min.
33

13. Centrifuge for 25 min at 13,000rpm at 4°C. Carefully remove the supernatant and add 500 µlof ice-cold 70%ethanol to the pellet.
14. Centrifuge for 10 min at 13,000 rpm at 4°C. Carefully remove the supernatant, leave tubes
open for 30 min at RT to evaporate the remaining ethanol.
15. Re-suspended the pellet in 20 µl of TE buffer.
16. Run samples (20 µl of DNA + 4 µl of 6x loading dye) in a 1.5% agarose gel.

From Plants to PCR results in 2 days!
Generally, the crosslinking of plant material and the chromatin extraction and sonication takes 1 to
1.5 days depending on the number and nature of samples. Another 0.5 to 1 day is needed to test theshearing efficiency (including 4h to overnight decrosslinking step). The chromatin
immunoprecipitation and DNA purification is generally done in 2 to 3 days depending on incubation
duration (4h or overnight). In total it can take from 3.5 to 5 days from Plant to results.
In some cases, when some conditions are fulfilled, it is possible to get PCR results from plants after
only 2 days (see below).

Conditions:
The user is well experienced with the kit.
The shearing conditions are already optimized for the plant material that will be used.
The experiment should be planned in advance (needed materials, consumables, calculation of
buffer and reagents volumes etc…).
This procedure may require long working days.

Planning:
DAY 1

34

•

Launch the antibody coating step (STEP 3. Magnetic Immunoprecipitation, 25-31).

•

During the incubation of antibody & DiaMag protein A-coated magnetic beads, crosslink the plantmaterial (STEP 1. Crosslinking of plant tissue) and proceed to the chromatin extraction andsonication (STEP 2. Chromatin extraction from plant tissue and chromatin shearing).

UNIVERSAL PLANT CHIP-SEQ MANUAL•During the sonication and the centrifugation step just after, the antibody coated magnetic beads
may be washed (STEP 3. Magnetic Immunoprecipitation, 32) and kept on ice.

•

When your chromatin is ready, proceed to the immunoprecipitation (STEP 3. Magnetic
Immunoprecipitation) Incubate chromatin with antibody-coated beads overnight.

DAY 2
•

Wash the beads (STEP 3. Magnetic Immunoprecipitation) and proceed to the elution and thedecrosslinking of DNA during 4 h (STEP 4. Elution, de-crosslinking and DNA isolation).

•

During the decrosslinking time, you can prepare the PCR plan and PCR mixes.

•

Purify the immunoprecipitated DNA (STEP 4. Elution, de-crosslinking and DNA isolation).

•

PCR can be launched at the end of day2 and results will be available the next day.

35

Related products
Product

Chromatin EasyShear Kit for Plant

C01020014

MicroPlex Library Preparation Kit v3 /48 rxns

C05010001

24 UDI for MicroPlex v3 - Set I

C05010008

IPure v2

C03010015

H3K27me3 Antibody

C15410195

ChIP/ChIP-seq grade antibodies
Bioruptor® Pico

36

Cat. No.

UNIVERSAL PLANT CHIP-SEQ MANUAL

www.diagenode.com
B01060010

Revision history
VersionDate of modification

Version 3 02_2024

February 2024

Description of modifications-

Addition of the Table 2.

-Update manual layout (the previous
one was last edited in 2017).
Table of content - update of reagents
versionRemoval of discontinued product(DiaMag 1.5 magnetic rack)
Addition of Tables: Related Products
and Revision history

Version 3 06_2023

June 2023
-

37

38

UNIVERSAL PLANT CHIP-SEQ MANUALFOR RESEARCH USE ONLY.
Not intended for any animal or human therapeutic or diagnostic use.
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transcribed, stored in retrieval systems, or translated into any language or computer language, in any formor by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without priorwritten permission from Diagenode SA (hereinafter, “Diagenode”). The information in this guide is subject tochange without notice. Diagenode and/or its affiliates reserve the right to change products and services at
any time to incorporate the latest technological developments. Although this guide has been prepared withevery precaution to ensure accuracy, Diagenode and/ or its affiliates assume no liability for any errors oromissions, nor for any damages resulting from the application or use of this information. Diagenode welcomes
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TRADEMARKSThe trademarks mentioned herein are the property of Diagenode or their respective owners. Bioanalyzer is atrademark of Agilent Technologies, Inc. Agencourt and AMPure® are registered trademarks of Beckman
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