Chromatin EasyShear Kit
for Plant
Previous name: Chromatin Shearing
Optimization Kit for Plant
Cat. No. C01020014

USER GUIDE
V3 04_2021

Please read this manual carefully
before starting your experimentSummary
Introduction

4

Kit Materials

5

Required Materials Not Provided

6

Kit Method Overview & Time Table

7

Remarks Before Starting

8

Short Protocol For Experienced Users

15

Detailed Protocol

19

STEP 1. Crosslinking Of Plant Tissue

20

STEP 2. Chromatin Extraction From Plant Tissue And Chromatin Shearing

23

STEP 3. Chromatin Shearing Analysis

27

Troubleshooting

29

Introduction
The first critical step of a successful Chromatin Immunoprecipitation(ChIP) experiment is the preparation of sheared chromatin. We therefore
suggest the use one of our optimized Shearing ChIP kits.
••••

Chromatin EasyShear Kit - Ultra Low SDS (Cat. No.C01020010)
Chromatin EasyShear Kit – Low SDS (Cat. No. C01020013)
Chromatin EasyShear Kit - High SDS (Cat. No.C01020012)
Chromatin EasyShear Kit for Plant (Cat. No.C01020014)

Our Chromatin EasyShear kits are used in combination with the Bioruptor  
in order to ensure a highly reproducible chromatin shearing and make sure
you obtain the right fragment size needed for your experiment. Establish
optimal conditions required for shearing cross-linked chromatin (proteinDNA) is usually laborious; the protocol of the Chromatin EasyShear kits isfast, easy-to-use and optimized to get the best results possible.The ChIP
assay offers great potential to improve knowledge about the regulationof gene expression. This technique is now used in a variety of life science
disciplines including cellular differentiation, tumor suppressor genesilencing, and the effect of histone modifications on gene expression.
Isolation and shearing of the chromatin after formaldehyde fixation is thefirst step in the ChIP method. The following two steps are: 1/ specificimmunoprecipitation of chromatin fragments using antibody against
the protein of interest and 2/ analysis of the immunoprecipitated DNA
fragment. With the present kit, the first step of the ChIP method is
guaranteed, providing you with the best results. Using standard protocols
and kits leads to a significant decrease in potential variables that canoccur from shearing to ChIP, which makes interpretation and analysis
from experiment to experiment much more accessible.

4

CHROMATIN EASYSHEAR KIT FOR PLANT MANUAL

Kit Materials
The content of the kit is sufficient to perform 12 chromatin extractions
and chromatin shearing optimization reactions. Store the components at
the indicated temperature upon receipt (Table 1).
Table 1: Components supplied with the kit
Description

Quantity

Storage

10x Crosslinking Buffer

36 ml

4°C

Crosslinking bags for plant tissue

15 pc

room temperature

Glycine

30 ml

4°C

4x Extraction buffer 1

90 ml

4°C

Extraction buffer 2

300 ml4°C

Extraction buffer 3

60 ml

4°C

Sonication Buffer

7.2 ml4°C

Elution Buffer 1

1.2 ml4°C, incubate at room
temperature before use

Elution Buffer 2

48 µl

4°C

DNA precipitant

120 µl4°C

DNA co-precipitant

60 µl-20 °CTE buffer

240 µl4°C5Required Material Not Provided
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•

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)
Bioruptor®
Thermomixer
Rotating wheel
Phenol/Chloroform/Isoamylalcohol {25:24:1}
Chloroform/Isoamylalcohol {24:1}
ß-Mercaptoethanol
Protease inhibitors (recommended: Protease Inhibitor Cocktail forplant cell and tissue extracts P 9599 from Sigma)• Pincer, Scalpel & blades, spatula etc…
• Towel papers

6

CHROMATIN EASYSHEAR KIT FOR PLANT MANUAL

Kit Method Overview & Time Table
Step

Time needed

STEP 1

Crosslinking of plant tissue

1 to 2 hours

STEP 2Chromatin extraction from plant tissue and chromatin shearing

2 to 5 hours

STEP 3Chromatin shearing analysis

1 day

7

Remarks Before Starting
Please read the complete manual very carefully before starting. It contains
many details which are very important and essential for the proper conductof experiments. We highly recommend using the detailed protocol for yourfirst assays. You can use the short protocol after being experienced withthis kit.
1. Starting material
This kit has been tested and validated for both monocots and dicots plants.
It was optimized for many model plant species belonging to many plantfamilies and several types of tissues (see table 2).
Depending on the plant tissue and/or species, the sampling method canbe different. In the majority of cases, we recommend cutting samples into
small pieces in order to facilitate the fixation with formaldehyde. 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 isno 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 then homogenise all organs (leaves, roots …) with apincer.
For tomato, cut young leaves into “discs” of about 4 to 5 mm using a blade
or sterile scissors.
8

CHROMATIN EASYSHEAR KIT FOR PLANT MANUAL

Tomato anthesis are sampled during the meiotic phase (buds between 3
to 6 mm). There is no need to 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 andmake a longitudinal “wound” along the stem using a sterile blade, thiswill facilitate the removal of the bark. Before removing the bark, cut thestem 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 describedfor 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 work on a completely different
tissue, the sampling procedure should be optimized.
The optimal weight of plant tissue for ChIP experiments depends on manyfactors:• 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 witha 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 to older
cells.
• Type of the cells: ex somatic cells vs sex organs• The genome size: plants with a bigger genome will yield morechromatin with fewer number of cells

9This protocol has been optimized for chromatin extraction from 0.1 to
2 g fresh weight (fw) of different plant materials (see examples in table2). It is possible to start with more than 2 g 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 chromatin extraction depending on theplant tissues
Optimalweight*Crosslinking
duration

Sonication(cycles)**

Centrifuge
speed ***

Arabidopsis seedlings
Arabidopsis thaliana ecotype columbia(col-0)1g

15 min81000 x g

Arabidopsis adult rosette leaves
Arabidopsis thaliana ecotype columbia(col-0)1g

15 min81000 x g

Tomato leaves
Solanum lycopersicum cv. Micro-Tom

1g

10

6-8

2100 x g

Tomato anthesis
Solanum lycopersicum

65 mg

10

6-8

4000 xg

Maize inner stem
Zea mays cv. B73

1.5 g

10

8-10

2900 x g

Maize husk ****
Zea mays cv. B73

1.5 g

10

8-10

2900 x g

Rice seedlings (2-3 leaves stage)
Oriza sativa ssp Japonica cv. Nipponbare

2g

15 min8-102900 x g

In vitro poplar plants (leaves + stem)
Populus trichocarpa clone Nisqually
Populus tremula x alba clone
INRA 717-1B4> 0.1 g

15 min82900 x g

Poplar (stem-differentiating xylem)
Populus trichocarpa clone Nisqually
Populus tremula x alba clone
INRA 717-1B4

2g

15 min102900 x g

Plant material* 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)
10

CHROMATIN EASYSHEAR KIT FOR PLANT MANUAL

*** 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 concentrationof formaldehyde is 3 %.

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 performedin vivo using fresh tissue in order to obtain the most accurate biologicalinformation relative to the research questions. This kit was therefore
optimized on fresh/living plant material. We note that it may be possible tocrosslink 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 ininefficient 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 a more efficient fixation. In fact, the cell wall may inhibit
the penetration of formaldehyde.
The Crosslinking buffer provided in this kit was optimized to yield morechromatin 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. Crosslinkingof 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 of material and will significantly
facilitate the crosslinking, the washes and sample handling after fixation.

11

Please note that provided crosslinking bags are not adapted to powder(ground plant material).
3. Shearing optimization and sheared chromatin analysis
Before starting the immunoprecipitation, 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 coolingsystem (#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 a different plant tissue(s) thesonication 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 optimalsonication conditions for your tissue. However, we highly recommend
optimizing the shearing conditions before doing any new experiment. In
fact, the shearing efficiency depends on numerous factors other than thesonication device and the plant material such as the sample volume, thechromatin 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 shortersonication 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
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.

12

CHROMATIN EASYSHEAR KIT FOR PLANT MANUAL

Figure 1
The figure shows gel electrephoresis of sheared chromatinof 3 independent nuclei preparations from maize innerstem. 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 thiswill result in reduced performance. Keep the beads in a homogenous
suspension at all times when pipetting. Variation in the amount of beads
will lead to lower reproducibility. Do not freeze the beads. The amount ofbeads needed per IP depends on the amount of antibody used for the IP.
The protocol below uses 20 μl of beads. The binding capacity of this amountis 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) included in this kit in this protocol (STEP4. Elution, decrosslinking and DNA isolation). The IPure kit v2 gives high quality DNA
for PCR and library preparation for next generation sequencing. It wasalso tested and validated for all plant tissues / or species indicated intable 2.
It’s highly recommended to quantify the eluted DNA using a Qubit for
example. This will give a first idea about the success of ChIP as therecovered DNA quantity should be different between IgG or no antibody
control (low), Input (high) and sample of interest (usually in between).

13

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 somecases. You can follow the manufacturer’s recommendations relative totemplate DNA concentration depending on your qPCR system and/or
reagents.
The efficiency of chromatin immunoprecipitation of particular genomic locican be expressed as the recovery of that locus calculated as the percentageof 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 a reference gene
(Normalized Enrichment). See below formulas for each type of calculation.
These equations assume that the PCR is 100 % efficient (amplification
efficiency = 2). For accurate results the real amplification efficiency, if
known, should be used.
NOTE: 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 have tendency to make dimers
resulting in a false peak (and as a consequence a false Ct value). In order toavoid this problem, use enough DNA for PCR. The Melting Peaks of IgG andsamples should be superposed with those of the Input (which generally has ahigher 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

14

CHROMATIN EASYSHEAR KIT FOR PLANT MANUAL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 apositive 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 arealso available 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 is provided 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.

15

Short Protocol For Experienced Users
STEP 1. Crosslinking of plant tissue
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 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 keepon 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 min.
6. Replace 2.5 ml of the crosslinking buffer by 2.5 ml of Glycine. Mix thesolution and apply vacuum for additional 5 min.
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 and snap-freeze in liquid nitrogen.

16

CHROMATIN EASYSHEAR KIT FOR PLANT MANUALSTEP 2. Chromatin extraction from plant tissue and chromatin shearing
9. Calculate the necessary amount of 4x Extraction buffer 1 (7.5 ml/sample),
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, Extraction buffer 2 and Extraction buffer 3 as indicated below
and keep them on ice.
Complete Extraction buffer 1
4x Extraction buffer 1

7.5 ml

dH2O

22.5 ml

Protease inhibitors

Add according to manufacturer’s instructions

ß-Mercaptoethanol

Add to a final concentration of 5 mM

Phenylmethylsulfonylfluoride (PMSF)

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 pre-cooled 50 ml tube.
12. Take the tubes out of the liquid nitrogen, wait 15 s and add 30 ml of cold
Complete Extraction Buffer 1.
1713. Incubate the tubes at 4°C in a rotating wheel for at least 15 min and up to
1 h.
14. Filter the suspension twice through 1 layer of Miracloth (Calbiochem) andcollect the flow-through in a new 50 ml tube on ice.
15. Centrifuge for 20 min at 4 °C at 1000 to 2900 x g (see table 2). Discard thesupernatant and avoid losing any part of the pellet.
16. Wash the pellet containing the nuclei 5 times with 5 ml Complete Extraction
Buffer 2. After each wash, centrifuge for 5 min at 4 °C at the same speed
used in the previous step and discard the supernatant.
17. Wash once with 5 ml Extraction Buffer 3 as in step 15. Centrifuge for 5 min
at 4 °C and discard the supernatant.
18. Centrifuge at 4 °C for 30s. Remove the rest of the extraction buffer 3 witha pipette.
19. Resuspend the pellet in 600 µl of Sonication Buffer (supplemented withprotease 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 min.
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 (seetable 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 min at 18.000 x g at 4 °C.
24. Transfer the supernatant to a new 1.5 ml tube.
NOTE: To identify correctly the fragment size distribution, sheared chromatinshould be decrosslinked (see step 3)

18

CHROMATIN EASYSHEAR KIT FOR PLANT MANUALSTEP 3. Magnetic 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 µl

dH2O1350 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 DiaMag1.5-magnetic rack. Wait 1 min and remove the supernatantby 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 the necessary amount (ov2) of washed beads (20 µl per ChIP
reaction for each Antibody / IgG). For each tube, if the final volume is lessthan 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 theoriginal volume (ov2) + 1 µl per ChIP reaction using 1x ChIP Dilution Buffer
and keep the tube on ice.

19

33. Per ChIP reaction: Dilute 40 µl of the sheared chromatin by adding 160 µlof cold 1x ChIP Dilution Buffer. Add protease inhibitors and mix thoroughly.
Place 2 µl of the diluted chromatin aside (-20 °C) to be used as INPUT later.
34. Per ChIP reaction, add 20 µl of Antibody coated magnetic beads to thechromatin. Incubate 4h to overnight in a rotating wheel at 4 °C.
35. After the incubation briefly spin the tubes and place them in the DiaMag1.5magnetic rack. Wait for one minute and remove the supernatant. Wash
beads once with 200 µl of wash buffer 1, 2, 3 and 4.

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 min at room temperature on
a rotating wheel.
37. Thaw your Input sample(s) from (step 3, 33) on ice.
38. After 30 min incubation, vortex, perform a short spin and put the strip on a
DiaMag02 magnetic rack and wait for 1 min.
39. Collect the supernatant, transfer it into a new 200 µl strip tubes. Add 98µl 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 4 h to overnightat 65 °C.
41. Add 2 μl of carrier to each IP and input sample. Vortex briefly and performa short spin.
42. Add 100 μl of 100% isopropanol to each IP and input sample. Vortex briefly
and perform a short 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).

20

CHROMATIN EASYSHEAR KIT FOR PLANT MANUAL

45. Prepare the Wash buffer 1 and 2 by adding 2 ml of Isopropanol (100 %) foreach.
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 a rotating wheel. Spin the strip, place it on a magnet anddiscard the supernatant.
48. Perform a second wash with 100 µl of wash buffer 2. Incubate for 30 s on arotating 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.

21

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.

22

CHROMATIN EASYSHEAR KIT FOR PLANT MANUALDETA I L ED
PR OTOCO L
STEP 1 - Crosslinking of plant tissue

20

STEP 2 - Chromatin extraction from plant tissue and chromatin shearing

23

STEP 3 - Chromatin shearing analysis

27

STEP 1
Crosslinking of plant tissue
WARNING: Steps 1.1, 1.4, 1.5, 1.6 and 1.7 should be carried out under
the fume hood

1.1

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

24

10x Crosslinking buffer

3 mlFormaldehyde

1 % (ex: 811 µl of Formaldehyde solution 37 %)dH2O

Up to 30 ml

Total volume

30 ml

1.2

Harvest the plant material (a description of the sampling procedure ofeach tested tissue and/or species is available in the section remarks
before starting, Starting material). Wash the plant material 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 direct contact with ice.

1.3

Weigh the necessary quantity of plant material mentioned in table 2 andstarting material section. If the goal is to optimise the shearing efficiency
or to compare many treatment conditions the weight should be exactly thesame for all samples in order to have reliable results. Put the weighted
sample in a crosslinking bag, make a node and pull simultaneously bothends of 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.

CHROMATIN EASYSHEAR KIT FOR PLANT MANUALIntroduce the closed crosslinking bag to the 50 ml falcon tube containing
the crosslinking solution supplemented with formaldehyde. Make surethat 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 the crosslinking bag.
This will somehow “lock” the crosslinking bag in the bottom of the tube
and prevent 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 the vacuum may induce variability.

1.5

Put the 50 ml falcon tubes containing samples in the desiccator (prefilled with ice). During the crosslinking under vacuum, the tubes have tobe 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.

1.6

Turn off the vacuum pump and release the vacuum slowly. Replace 2.5 mlof the crosslinking buffer by 2.5 ml of Glycine. Mix the solution and apply
vacuum for additional 5 min. Release the vacuum slowly.

1.7

Discard the Crosslinking Buffer following the legislation relative todangerous chemical products of your country. Add 40 ml of cold deionizedwater (4 °C; not supplied), close the tube and mix. Discard the water withdangerous products as it may contain formaldehyde. Repeat the washing
step twice (3 washes in total).

1.8

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 andsnap-freeze them in liquid nitrogen. At this point the tissue can be stored
at - 80 °C for several months or directly used for chromatin extraction.

PROTOCOL

1.4

25

NOTE: For poplar plants grown in greenhouse and/or field, remove the barkfrom stem fragments. Place a clean box (ex: clean empty tips box) on ice inthe desiccator and fill it with stem fragments. Add the necessary 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. Releasethe vacuum slowly.
Discard the Crosslinking Buffer and wash the stem fragments 3 times
generously with cold deionized water. Place the fragments on towel paper.
Scrape gently the outermost layer of stem fragments corresponding to thestem-differentiating xylem (SDX) using a blade. The scraped SDX should becollected in a clean recipient containing liquid Nitrogen until the end of thesampling. Weigh the SDX and store in aliquots of 2 g each at -80°C up to several
months.

26

CHROMATIN EASYSHEAR KIT FOR PLANT MANUAL

Chromatin extraction from planttissue and chromatin shearing

PROTOCOL

STEP 2WARNING: Buffers containing ß-mercaptoethanol should be prepared, handled
and discarded under the fume hood!

2.1

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

2.2

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 ml

dH2O

22.5 ml

Protease inhibitors

Add according to manufacturer’s
instructions

ß-Mercaptoethanol

Add to a final concentration of 5 mM

Phenylmethylsulfonylfluoride (PMSF)

Add to a final concentration of 0.1 mM

Total volume

30 ml

Complete Extraction buffer 2(Volume for 1 sample)

4x 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
27

28

Complete Extraction buffer 3(Volume for 1 sample)

4x 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

25 ml

2.3

Fill a ceramic mortar with liquid nitrogen and add the plant tissue. Grindthe plant tissue thoroughly using a mortar and a pestle to a fine powder
in liquid nitrogen. Avoid letting the tissue to thaw at any time. Transfer the
powder to an empty pre-cooled 50 ml tube. As it is 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 thetube and keep it in liquid Nitrogen. Repeat this procedure for the othersamples.

2.4

When all samples are grinded, take the tubes out of the liquid nitrogen,wait 15 s and add 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.

2.5

Incubate the tubes at 4°C in a rotating wheel for at least 15 min 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 and nuclei.

2.6

Filter the suspension twice through 1 layer of Miracloth (Calbiochem)and collect the flow-through in a new 50 ml tube on ice. The pore size ofMiracloth layer is about 22 µm. The nuclei having around 10 µm diameter
will pass through the pores but not the bigger cell lysates.

2.7

Centrifuge for 20 min at 4 °C at 1000 to 2900 x g depending on plantmaterial (see table 2). The nuclei being heavier than the remaining celllysates will remain in the bottom of the falcon tube after centrifugation.
Discard the supernatant and avoid losing any part of the pellet.

CHROMATIN EASYSHEAR KIT FOR PLANT MANUALWash the pellet containing the nuclei 5 times with 5 ml Complete
Extraction Buffer 2. In each washing step carefully resuspend the pellet
by gentle shaking (no vortexing or pipetting). The pellet may be difficult toresuspend. Thus alternate short shaking with incubation on ice for 15s.
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 min at 4 °C at the same speed used in the previous stepand discard the supernatant without disturbing the pellet.

2.9

Wash once with 5 ml Extraction Buffer 3 as in step 15. Centrifuge for 5
min at 4 °C and discard the supernatant.

PROTOCOL

2.8

2.10 Centrifuge at 4 °C for 30s. Remove very carefully the rest of the extraction
buffer 3 with a pipette. Remove only the clear liquid without disturbing
the pellet. If the liquid is not clear you can centrifuge again for 30s. It is
important to remove all the remaining liquid before adding the sonicationbuffer. Keep the tubes with the pellets on ice. At this step, the nuclei
pellet can be stored at - 80 °C.
2.11 Resuspend the crude nuclear pellet in 600 µl of Sonication Buffer(supplemented with protease inhibitors) by pipetting and transfer thesuspension 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.
2.12 Incubate the 1.5 ml tube at 4°C in a rotating wheel for 15 min. During thisstep, the sonication buffer will permit the lysis of the nuclear membrane
before sonication making the chromatin more accesible for sonication.
2.13 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 manysonication tubes as necessary.

29

2.14 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 thetissue type, fixation time and Bioruptor® model used. We recommend
performing a time course experiment in order to specify the optimalsonication conditions (see Remarks before starting).
2.15 Transfer the sonicated chromatin to a standard 1.5 ml tube and centrifuge
for 5 min at 18.000 x g at 4 °C.
2.16 Transfer the supernatant to a new 1.5 ml tube. We recommend highly
measuring the concentration 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 to identify the chromatinyield 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).
A calculation table is available for downloading in Diagenode website(same location as the manual)(N) = (0.978 x Q x V x 10^6) / Gs(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
At this point the chromatin can be stored at - 80 °C or directly used for the
immunoprecipitation. Avoid multiple freeze/thaw cycles.
NOTE: To identify correctly the fragment size distribution, sheared chromatinshould be decrosslinked (see step 3)
NOTE: Not sheared chromatin from step 20 can also be stored at -80°C andsheared after thawing

30

CHROMATIN EASYSHEAR KIT FOR PLANT MANUAL

Chromatin shearing analysis
3.1

Take an aliquot of 50 μl of sheared chromatin and spin the chromatin at
12,000 rpm for 10 min at 4°C. Transfer the supernatant to a new tube forchromatin analysis.

3.2

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

3.3

Add 2 μl of diluted RNase cocktail.

3.4

Incubate 1h at 37°C.

3.5

Add 50 μl of elution buffer 1.

3.6

Add 4 μl of elution buffer 2, mix thoroughly.

3.7

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

3.8

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.

3.9

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

PROTOCOL

STEP 33.10 Add 1 volume of chloroform/isoamyl alcohol (24:1). Incubate the sample
at RT for 10 min on a rotating wheel.

31

3.11 Centrifuge for 2 min at 13,000 rpm at RT. Transfer the top aqueous phase
into a new 1.5 ml tube.
3.12 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.
3.13 Centrifuge for 25 min at 13,000rpm at 4°C. Carefully remove thesupernatant and add 500 μl of ice-cold 70% ethanol to the pellet.
3.14 Centrifuge for 10 min at 13,000 rpm at 4°C. Carefully remove thesupernatant, leave tubes open for 30 min at RT to evaporate the remaining
ethanol.
3.15 Re-suspended the pellet in 20 μl of TE buffer.
3.16 Run samples (20 μl of DNA + 4 μl of 6x loading dye) in a 1.5% agarose gel.

32

CHROMATIN EASYSHEAR KIT FOR PLANT MANUALTroubleshooting
If you face an issue not cited below or if any of the proposed solutions
don’t help resolving an issue, please contact Diagenode
Issue

Possible solution(s)Non-efficient
shearing(abundant fragmentswith high size
when analyzing theshearing efficiency)• Re-sonicate chromatin 2 to 6 additional cycles (30s ON/30s OFF)on Bioruptor Pico and analyze the shearing efficiency.
• Reduce concentration of chromatin (dilute in sonication buffer)• Reduce crosslinking time (over-crosslinking may reduce shearing
efficiency).
• A de-crosslinking step should be included when analyzing theshearing efficiency• Check all sonication parameters related to Bioruptor(recommended tubes, volumes …). Please visit Diagenode website

Low chromatin yield• Grind well the plant material to a fine powder• Increase quantity of starting material• Increase incubation time (step 13 and step 20)• Remove filtration step with Miracloth especially with very small
quantity of starting material or rare samples (step 14)• Reduce the volume of sonication buffer used to take the nuclei
pellet (ex: 200-300 µl instead of 600 µl) (step 19)

33

FOR RESEARCH USE ONLY.
Not intended for any animal or human therapeutic or diagnostic use.
© 2021 Diagenode SA. All rights reserved. No part of this publication may be reproduced, transmitted,
transcribed, stored in retrieval systems, or translated into any language or computer language, in any
form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise,
without prior written permission from Diagenode SA (hereinafter, “Diagenode”) . The informationin this guide is subject to change without notice. Diagenode and/or its affiliates reserve the rightto change products and services at any time to incorporate the latest technological developments.
Although this guide has been prepared with every precaution to ensure accuracy, Diagenodeand/or its affiliates assume no liability for any errors or omissions, nor for any damages resulting
from the application or use ofthis information. Diagenode welcomes customer input on corrections
and suggestions for improvement.
NOTICE TO PURCHASER LIMITED LICENSE
The information provided herein is owned by Diagenode and/or its affiliates. Subject to the terms andconditions that govern your use of such products and information, Diagenode and/or its affiliates
grant you a nonexclusive, nontransferable, non-sublicensable license to use such products andinformation only in accordance with the manuals and written instructions provided by Diagenodeand/or its affiliates. You understand and agree that exceptas expressly set forth in the terms andconditions governing your use of such products, that no right or license to any patent or otherintellectual property owned or licensable by Diagenode and/or its affiliates is conveyed or implied
by providing these products. In particular, no right or license is conveyed or implied to use theseproducts in combination with any product not provided or licensed to you by Diagenode and/or its
affiliates for such use. Limited Use Label License: Research Use Only The purchase of this productconveys to the purchaser the limited, non-transferable right to use the product only to perform
internal research for the sole benefit of the purchaser. No right to resell this product or any of
its components is conveyed expressly, by implication, or by estoppel. This productis for internal
research purposes only and is not for use in commercial applications of any kind, including, without
limitation, quality control and commercial services such as reporting the results of purchaser’s
activities for a fee or other form of consideration. For information on obtaining additional rights,
please contact info@diagenode.com.
TRADEMARKSThe trademarks mentioned herein are the property of Diagenode or their respective owners.
Bioanalyzer is a trademark of Agilent Technologies, Inc. Agencourt and AMPure® are registeredtrademarks of Beckman Coulter, Inc. Illumina® is a registered trademark of Illumina®Inc; Qubit is a
registered trademark of Life Technologies Corporation.

www.diagenode.com