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introDuctionAngiosperm seeds contain a nourishing endosperm tissue that regulates nutrient transfer to the developing embryo, performing a functional role similar to that of the placenta in mammals. Embryo and endosperm are both products of a double fertilization event in which the two haploid male gametes (sperm cells) fuse with two female gametes, the haploid egg cell and the homodiploid central cell1. As the central cell is diploid, the resulting endosperm is a triploid tissue containing two maternal genomes and one paternal genome. To understand the epigenetic processes balancing maternal and paternal genome contributions in the endosperm, parental-specific epigenome studies are a fundamental requirement. However, in most dicot species, the early endosperm is a syncytium and consists of only a few hundred nuclei that are difficult to extract using manual dis-section. Therefore, the adaptation of the INTACT (for isolation of nuclei tagged in specific cell types) technique2 for purify-ing endosperm nuclei is a major technical advance that previ-ously allowed us to establish the specificities of parental-specific chromatin modifications in the early endosperm3.

The INTACT method was developed a few years ago2 and was used for the specific purification of nuclei from Arabidopsis root tissues. The method is based on cell-type-specific labeling of nuclei with a GFP fused to a nuclear targeting fusion protein (NTF) that can be biotinylated by the Escherichia coli biotin ligase (BirA), permitting the purification of labeled nuclei using streptavidin-coated magnetic beads. We established a transgenic Arabidopsis line (named INT) expressing the NTF and BirA components of the INTACT system4 under the endosperm-specific PHERES1 (PHE1) promoter5 that is active until ~4 d after pollination (DAP)6. Using this purification method, we have been able to isolate endosperm nuclei at 4 DAP without substantial contamination from the embryo and the surrounding maternally derived seed coat and silique tissues. Purified nuclei were used for either the analysis of

histone modifications by ChIP or DNA methylation by BS-seq. Using the Arabidopsis accessions Columbia (Col-0, referred to hereafter as Col) and Landsberg erecta (Ler) as maternal and paternal parents that differ in high numbers of SNPs7,8, we were able to determine the parental origin of these modifications. The procedure presented here (Fig. 1) allows the user to create high-quality, cell-type-specific and parent-of-origin-specific epigenome profiles from purified endosperm nuclei. We also provide com-plete instructions for both ChIP and bisulfite treatment followed by next-generation sequencing (ChIP-seq and BS-seq, respec-tively). Detailed instructions on how to optimize the INTACT protocol are provided, allowing adaptation of this method to other tissues (Boxes 1 and 2). Finally, we provide detailed instruc-tions on reliable purity assessments making use of either spike-in material or distributions of parental sequence polymorphisms (Box 3; Supplementary Notes 1–3), avoiding microscopic analysis or RNA-expression-based purity quantifications.

Development of the protocolInitially, we carried out INTACT purification of endosperm nuclei by following the protocol established to purify root nuclei4. However, the isolated nuclei were highly contaminated by non-endosperm nuclei, indicating the need for additional optimiza-tion steps to adapt the protocol to endosperm tissue.

We focused on four points that we consider to be critical to successfully adapt the procedure. (i) Obtaining of endosperm- specific INTACT lines with high levels of NTF and BirA expression: the presence of GFP in the NTF construct allowed us to select for lines with high expression levels and corroborate the endosperm specificity of the PHE1 promoter that is specifically expressed in endosperm nuclei at 1–5 DAP9 (Fig. 2a). The efficiency of the BirA activity in the different lines was assessed by western blot-ting (Fig. 2b) according to previously published procedures2.

Applying the INTACT method to purify endosperm nuclei and to generate parental-specific epigenome profilesJordi Moreno-Romero, Juan Santos-González, Lars Hennig & Claudia Köhler

Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden. Correspondence should be addressed to J.M.-R. (Jordi.moreno@slu.se) or C.K. (Claudia.Kohler@slu.se).

Published online 5 January 2017; doi:10.1038/nprot.2016.167This protocol is an extension to Nat. Protoc. 6, 56–68 (2011); doi:10.1038/nprot.2010.175; published online 16 December 2010

the early endosperm tissue of dicot species is very difficult to isolate by manual dissection. this protocol details how to apply the intact (isolation of nuclei tagged in specific cell types) system for isolating early endosperm nuclei of Arabidopsis at high purity and how to generate parental-specific epigenome profiles. as a protocol extension, this article describes an adaptation of an existing nature protocol that details the use of the intact method for purification of root nuclei. We address how to obtain the INTACT lines, generate the starting material and purify the nuclei. We describe a method that allows purity assessment, which has not been previously addressed. the purified nuclei can be used for chip and Dna bisulfite treatment followed by next-generation sequencing (seq) to study histone modifications and Dna methylation profiles, respectively. By using two different Arabidopsis accessions as parents that differ by a large number of single-nucleotide polymorphisms (snps), we were able to distinguish the parental origin of epigenetic modifications. our protocol describes the only working method to our knowledge for generating parental-specific epigenome profiles of the early Arabidopsis endosperm. the complete protocol, from silique collection to finished libraries, can be completed in 2 d for bisulfite-seq (Bs-seq) and 3 to 4 d for chip-seq experiments.

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To ensure that the biotinylation level of the NTF nuclei was suf-ficient for bead binding, we used a binding assay (Box 1; Fig. 2c). (ii) Optimization of the tissue homogenization and nuclei extraction method is required for each tissue (Box 2; Fig. 3a,b): the NTF fluorescence allows the researcher to test the integrity of the isolated nuclei (Fig. 3c) and to decide on the best extraction method. (iii) Assessment of purity and optimization of yield: a proper assessment of nuclei purity is critical to optimizing the protocol. We therefore developed a quantitative-PCR (qPCR)-based method with spike-in material of plants not expressing the INTACT constructs (Box 3). (iv) Optimization of ChIP and bisulfite procedures to allow preparation of sequencing libraries: after these four optimization steps, we were able to purify early endosperm nuclei to high purity, perform ChIP-seq and BS-seq, and obtain parental-specific epigenome profiles3.

Comparison with other endosperm isolation methodsPrevious genome-wide studies of the transcriptome or epigenome of the Arabidopsis endosperm were based on manual endosperm dissection10–13, laser capture microdissection (LCM)8,14 or FACS9,15. In the following section, we will compare those methods with the INTACT method. Endosperm purification by manual dissection is a highly laborious method that is feasible only at later stages of endosperm development (starting at ~6 DAP). Manual dissection has been previously used to collect tissue for the generation of DNA methylation profiles10–13 and transcriptome profiles8,16,17. Because of the high amount of

starting material required for ChIP experiments, manual dissec-tion is not a suitable approach. For the same reason, LCM has been applied only to isolate tissue for the generation of transcriptome profiles8,14. Epigenome profiles of the early endosperm have been generated using FACS-purified endosperm nuclei that have been GFP-labeled using the PHE1 promoter9,15. Nevertheless, as FACS requires high amounts of starting plant material, this method is not suitable for generating parental-specific epigenome profiles that use limited numbers of hand-pollinated siliques as start-ing material. Furthermore, the INTACT method does not rely on expensive flow cytometry facilities, making it a superior method as compared with FACS-based methods.

Comparison of INTACT protocolsAside from changes to the original protocol concerning nuclei extraction (described in Boxes 1 and 2), the main advance of this protocol is a rigorous purity assessment that forms the basis for successful protocol optimization. Preceding protocols have assessed purity either indirectly by gene expression analysis4,18 or directly by determining the number of labeled (NTF-positive) and unlabeled (NTF-negative) nuclei2,4. Using gene expression as a method to determine purity has the disadvantage that it relies on knowledge of tissue-specific gene expression and is very difficult to quantify. In addition, determining the ratio of NTF-positive and NTF-negative nuclei as an assessment of purity heavily underestimates the level of contamination. When we estimated the purity of isolated endosperm based on the relative number of NTF-positive and NTF-negative nuclei, we calculated contamination levels of 3 and 6%, respectively, in each of the replicates. However, after sequencing the DNA of the isolated nuclei, we calculated contamination levels of 27% and 38%, respectively, based on the deviation from the expected 2:1 maternal/paternal genome ratio in the endosperm (first repli-cate: 5,362,482 maternal and 1,272,540 paternal reads; second replicate: 7,893,140 maternal and 1,374,402 paternal reads; con-tamination calculated as detailed in Box 3, Formula 3). Using a qPCR-based purity assay (Box 3), we could estimate the Col and Ler ratios before and after purification, either by using spike-in material of the Ler accession or using material of Col and Ler inter-accession crosses. Evaluating the purity using that assay, we were able to optimize all steps of the procedure. We observed a major improvement of nuclei purity when including BSA in the incuba-tion step with streptavidin beads, causing a marked reduction of unspecific chromatin binding to the Dynabeads. Contamination levels substantially increased when cross-linked material was used; although the binding to the beads was not compromised, the yield of purified nuclei decreased and the contamination heavily increased (Supplementary Fig. 1). For that reason, we performed the cross-linking after nuclei purification.

LimitationsThe INT line expresses the NTF and BirA components under the endosperm-specific PHE1 promoter. As PHE1 activity strongly declines at 5 DAP, the latest stage of endosperm development that can be analyzed using this protocol is at 4 DAP. Earlier time points have not been tested, but they are expected to increase the contamination level because of the reduced numbers of endosperm nuclei as compared with those of nonendosperm nuclei from surrounding maternal tissues. This may require

Histone modificationprofiles

DNAmet profiles

Tissue preparation and collection

Steps 1–4

Nuclei extraction

Purification of labeled nuclei

BS-seq librarypreparation

ChIP-seq library preparation

Steps 5–17

Bindingassay

Purityassay

Steps (i–iv)

Emasculation–pollinationSilique collection

Final puritycalculation

(Step 27A) Bisulfitesequencing

(Step 27B) ChIP

qPCRChIP

Tissue homogenizationNuclei extraction

Streptavidin bead bindingLabeled nuclei purification

Nuclei cross-linkingChromatin shearing

Chromatin IP

DNA de-cross-linking

DNA purification and qPCR

DNA purification

DNA shearing

Sequencing

6 d

30 min

Steps 18–26 30 min

60 min

Steps (i–x)

Steps (vi–x) 5–9 h Steps (xxx–xxxii) 3–5 h

Box 3 2 h

40 min + O/N

Steps (xi–xxiii) 2 h + O/N

Chromatin IP washes

Steps (xxiv–xxvii) 3 h

Box 3

Box 1 1 h

Parental-specific epigenetic profiles

Figure 1 | Experimental workflow and time estimates. Steps (in blue) and time (in red) for the complete procedure. DNAmet, DNA methylation; O/N, overnight.

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longer washes and increased amounts of starting material. It also must be considered that, in some mutants, PHE expression can be affected; for that reason, it is important to test expression on the INTACT components in advance of an experiment involving mutants (Box 1).

Experimental designCloning. For our previous study, we generated independent transgenic lines by transforming Arabidopsis Col plants with the

pPHE1::NTF and pPHE1::BirA constructs using Agrobacterium-mediated transformation19. Plasmids were generated based on published constructs2,4 using BP/LR Gateway cloning technology (Invitrogen). The NTF fragment was amplified using the primers NTF-attB1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAATCATTCAGCGAAA-3′) and NTF-attB2 (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTACATCTAGTAACATAGATG-3′) and the BirA fragment was amplified using BirA-attB1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAAG

Box 1 | Obtaining INTACT lines and the nuclei-binding assay ● tiMinG 2 h The nuclei binding assay is an easy and fast way to select the best INTACT lines (different NTF and BirA combinations; Fig. 4). The amount of starting material required for this assay is less than for the whole procedure described, but it should be sufficient to identify positive nuclei under the microscope. Different homogenization protocols can be used (Box 2). All steps are performed on ice or in a cold room. Put buffers on ice and cool down the centrifuges. Avoid pipetting nuclei extracts excessively and always use cut tips when pipetting. It is useful to test the integrity of the nuclei using DAPI staining. For concentrating the nuclei suspension, centrifugation at 1,500g for 4–6 min at 4 °C can be used.

Material collection ● tiMinG 20 min1. Test the endosperm fluorescence signal of ~4 DAP seeds under the microscope (Fig. 2a).2. Collect the material (follow main PROCEDURE Steps 1–4). The amount of material required is 100 mg of siliques or less.

tissue homogenization and nuclei extraction ● tiMinG 40 min3. Place the frozen siliques from the liquid N2 in a precooled mortar. critical step Avoid thawing the tissue, and be aware that when the N2 inside the siliques evaporates, they can explode.4. Break the siliques with a precooled pestle. Do not apply a lot of pressure to the tissue; the endosperm is a syncytial tissue that does not need excessive grinding to be broken. critical step Grind with the pestle once the N2 is evaporated, and avoid adding extra N2 to prevent breakage of nuclei.5. Add the obtained powder to a 50-ml tube with 3 ml of nuclei extraction buffer. Avoid freezing the solution. critical step This step is crucial for nuclei integrity; freezing will destroy nuclei. critical step Alternatively, other nuclei extraction methods can be used (see PROCEDURE for GentleMACS dissociation and Box 2 for the chopping protocol).6. Incubate under gentle rotation for 15 min at 4 °C.7. Filter the homogenate through one layer of Miracloth into a 15-ml tube.8. Wash the 50-ml tube with an extra 1 ml of nuclei extraction buffer and filter in the same 15-ml tube from step 7.9. Filter the homogenate through a CellTrics strainer or two layers of Miracloth.10. Centrifuge at 1,500g for 5 min at 4 °C.11. Remove the supernatant and resuspend the pellet in 150 µl of PBS using a fine brush. Touch the pellet gently with the tip of the brush to break it, and afterward break the pieces of pellet until a homogeneous resuspension is achieved. Transfer the mixture to a 1.5-ml tube.

Determination of nuclei integrity and positive nuclei detection ● tiMinG 20 min12. Take a sample (10–15 µl) from the resuspended nuclei extraction.13. Add DAPI (final concentration 1 µg/ml) and analyze the sample under the fluorescence microscope. The nuclei are visible under a 40–60× magnification objective, but 100× is useful for detecting the NTF fluorescence in the nuclear envelope. Test for nuclei integrity and count GFP-positive nuclei and negative nuclei (Fig. 3c).? trouBlesHootinG

Binding assay ● tiMinG 40 min14. Wash 20 µl of M-280 streptavidin Dynabeads twice with 50 µl of PBS using a magnetic rack. BSA incubation as in the main protocol is not needed in this optimization step.15. Add 20 µl of the washed M-280 streptavidin Dynabeads to the resuspended nuclei (Step 11).16. Incubate for 30 min at 4 °C with gentle rotation.17. Collect the Dynabeads with the magnetic rack. Take an aliquot, add DAPI and test under the microscope for the presence of nuclei bound to beads (Figs. 2b and 3a). After using the magnet, the positive nuclei will have more beads attached (due to the increase in bead concentration when you capture the beads with the magnet) and it will be easier to detect clusters of beads surrounding the nuclei (Figs. 2b and 3a). critical step Wash the beads two to three times with PBS to reduce the amount of negative nuclei in the sample; this will help to identify positive nuclei covered by beads under the microscope. If this step is performed, skip the next step (step 18).18. Continue bead incubation (repeat steps 16 and 17) if nuclei coated with beads have not been identified.

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GATAACACCGTG-3′) and BirA-attB2 (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTACGACGGGGATCTGGATTT-3′). The fragments were introduced into pDNOR221 and transferred into the pB7WG2 pDEST vector containing a 3-kb PHE1 promoter

sequence. Transgenic lines with one insertion event were selected based on segregation analysis. Two NTF lines with specific and high fluorescence in the endosperm were crossed with different BirA lines. Lines homozygous for both constructs were selected

Box 2 | Nuclei extraction methods ● tiMinG variableThe success of nuclei extraction depends on the quality of the starting material and the homogenization procedure. Each tissue requires a proper extraction method that balances the low yield of nuclei obtained using gentle extraction procedures with the breakage of nuclei that occurs with harsh homogenization methods.

chopping ● tiMinG 15–20 minThis method is a fast and easy procedure, and it allows a gentle nuclei release from the tissue. Although this method keeps most nuclei intact, its efficiency is very low and it is thus suitable only when few nuclei are required for analysis.

1. Place the siliques (or other tissue in case you want to test other INTACT lines) in a plastic Petri dish on ice.2. Add 200 µl of PBS and chop the tissue with a scalpel blade.3. Slowly pipette up and down five to six times with a 1-ml cut pipette tip.4. Filter with a CellTrics strainer and test the presence of nuclei under the microscope using DAPI staining (1 µg/ml final concentration).

n2 grinding ● tiMinG 40 min This method provides a very efficient nuclei release, but the integrity of many nuclei is compromised. Differences in the grinding pressure have an impact on extraction efficiency and nuclei integrity, impairing reproducibility of this method. We also observed contamination problems caused by small tissue pieces (Fig. 3a,b). Nevertheless, when properly adjusted, this method can be used for many tissues. The protocol for extracting using N2 grinding is described in Box 1.

GentleMacs dissociator ● tiMinG 30 min To avoid the contamination problems associated within the N2 grinding method and to maintain nuclei quality, it is advisable to choose a tissue homogenization method that releases nuclei directly to the extraction buffer. The GentleMACS dissociator system is used in the main PROCEDURE, although rotor homogenizers (rotating blades) or dounce homogenizers can be possible alternatives to be tested. The use of GentleMACS reproducibly results in high numbers of intact nuclei and only few tissue fragments in the extraction (Fig. 3). The homogenization protocol using GentleMACS is described in the main PROCEDURE (Steps 5–16).

Box 3 | Purity assay ● tiMinG 2 hEndosperm parental-specific chromatin profiles require highly pure material, as any contamination will introduce parental bias, mostly from the maternal silique tissues. We use Col and Ler sequence polymorphisms to determine purity. We either calculate deviations from the expected 2:1 maternal/paternal genome ratio when using hybrid seeds derived from Col × Ler crosses or add spike-in of Ler silique material (20–30% (wt/wt) of the total sample) before homogenization of the Col silique material. We have developed a purity assay to quantify contamination by qPCR that is helpful as a control either before sequencing library preparation or for optimization of the protocol steps. After sequencing, contamination can be determined based on the assignment of sequencing reads to maternal and paternal alleles, and calculation of deviations from the expected ratio. For calculation examples, see supplementary notes 1 and 2.

purity assay1. Prepare DNA reference samples containing Col and Ler genomic DNA in the following ratios: 1:1 (ref1:1), 1:0 (ref1:0) and 0:1 (ref0:1). critical step Prepare Col:Ler reference samples in 1:2 and 2:1 ratios. In a Col × Ler cross, the Col:Ler ratio (RC:L) obtained should be the same as in ref2:1 (RC:L = 0.33); in a Ler × Col cross, the value should match with that of ref1:2 (RC:L = 0.66). Optionally, use the NPC control sample (Step 17). Purify the DNA following standard phenol/chloroform extraction with ethanol precipitation.2. Take the purified DNA from the BS-seq protocol (from Step 27A(v)) or the input sample from ChIP (from Step 27B(xxviii)) and dilute 1:20 (vol/vol) in water.3. The purity assay is performed by qPCR-amplifying a Ler-specific DNA fragment using primers for Ta1-2 and comparing the quantity of this fragment with a common PCR product of Col-0 and Ler using primers for ACT7. Perform qPCR with the purified sample and the DNA references using the ACT7 and Ta1-2 primers. The sample ref1:0 should be positive for ACT7 and negative for Ta1-2. The sample ref0:1 should be positive for both. Run the reactions on a qPCR machine with the following program:

Cycle no. Denature (98 °C) Anneal (65 °C) Extend (72 °C) Hold (4 °C)

1 30 s 2–22 10 s 30 s 30 s 23 5 min 24

? trouBlesHootinG(continued)

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Box 3 | Purity assay ● tiMinG 2 h (continued)4. Analyze the cycle threshold (Ct) values, following option A for Col and Ler interaccession crosses and option B for Col × Col crosses with a Ler spike-in.

(a) col and ler interaccession crosses(i) Calculate as follows (see Example 1, supplementary note 1): determine the Ler ratio (RL), calculated as:Formula 1

RL

Ct T ref

Ct ACT ref= ⋅

−

−2

20 5 1

1 1 1

7 1 1

∆

∆

, ( , :

, ( , :. (

a sample)

sample)))

where ref,1:1 is the Ct obtained in the ref1:1 reference DNA and sample refers to the purified DNA. The RL is multiplied by 0.5 because the reference ref1:1 is used. critical step Optionally, you can use other reference DNA combinations as a control. If Col:Ler reference DNA ref0:1 is used as ref, then RL should not be multiplied by 0.5 (as Ler is 100% (wt/wt)).(ii) The RL is used to calculate the fraction of paternal DNA (Rpat) that depends on the direction of the cross: Rpat = RL in Col × Ler crosses and Rpat = 1 – RL in Ler × Col crosses. The Rpat value is used to calculate the ratio of maternal:paternal (Rmat:pat) DNA as follows:Formula 2

RR

Rmat patpat

pat. ( )=

−12

(iii) Finally, Rmat:pat will be used to calculate the contamination:Formula 3

Contamination =+

=⋅ −⋅ +

NE

E NE

R

Rp

p p

mat pat

mat pat

1 2 1

1 2 23

/

/( ).

.

where Ep is the number of endosperm nuclei after purification and NEp is the number of nonendosperm nuclei after purification. The formula is obtained considering Rmat:pat = maternal alleles/paternal alleles, maternal alleles = 2/3·Ep + 2NEp, and paternal alleles = 1/3·Ep. For a detailed derivation of Formula 3 see supplementary note 2.(B) col × col crosses with ler spike-in(i) Calculate as follows (see Example 2, supplementary note 1): this calculation requires determining the ratio of INTACT positive and negative nuclei in your homogenized sample before purification (Enpc, Step 17). Calculate the spike-in (S) in the NPC sample (Snpc) and in the purified sample (Sp):Formula 4

SCt T ref

Ct ACT ref= ⋅

−

−2

20 5 4

1 1 1

7 1 1

∆

∆

, ( , :

, ( , :. ( )

a sample)

sample)

Snpc can be also calculated as weight of Ler siliques spiked-in/weight of Col siliques.(ii) Calculate the contamination considering that the ratio of nonendosperm nuclei (NE) and spike-in nuclei (S) before and after purification should be the same:

NE

S

NE

Sp

p

npc

npc=

Use the following formula to calculate the contamination:Formula 5

Contamination = + = ⋅ −S NES

SEp p

p

npcnpc( ) ( )1 5

Final contaminationAfter sequencing, reads can be assigned to the Col and Ler genomes, which allows for the final contamination of the sample. We did not observe substantial differences of contamination levels in reciprocal Col × Ler and Ler × Col crosses3, indicating that the expected reduced mapping efficiency of Ler reads to the Col reference genome by SNPsplit introduces negligible bias. In addition, the SNPsplit method assignment of Col and Ler reads has a technical error (errorSNPsplit) of 0.013 ± 0.011 (calculated using Formula 5), based on six experiments of Col tissue without Ler spike-in. This value can be subtracted from the final contamination calculation.

• In Col and Ler interaccession crosses (see Example 1, supplementary note 3): use Formula 3 to calculate the contamination. To calculate Rmat:pat, we use the number of maternal reads (rmat) and the number of paternal reads (rpat) using Rmat:pat = rmat/rpat.

• In Col × Col crosses with Ler spike-in (see Example 2, supplementary note 3) use Formula 5. To calculate Sp, use the number of Ler reads (rler) and number of Col reads (rcol) using Sp = rler/rcol. ? trouBlesHootinG

(1)(1)

(2)(2)

(3)(3)

(4)(4)

(5)(5)

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based on fluorescence and PCR genotyping (using specific prim-ers for the BirA construct (5′-CACATGCCTACACGTAAG-3′ and 5′-GCTACACTACCGCCATCC-3′)). We selected for lines express-ing high levels of biotinylated NTF (Fig. 1b) by western blotting and bead-binding assay (Box 1; Fig. 1b). One line, named Col INT, was used for the experiments and introgressed into the condi-tional male sterile mutant delayed dehiscence 2 (dde2)20.

Crosses. To generate parental-specific epigenetic profiles, Col INT plants (in the Col accession background) can be used as maternal or paternal parents crossed with Ler plants (Fig. 4). To ease the workload for crosses, the dde2 mutant or pistil-lata (pi-1)21 can be used as the female parent, allowing for the omission of emasculation.

Biotinylation efficiency. The efficiency of biotinylation can be assessed using the bead-binding assay (Fig. 4). Purity of endosperm nuclei is assessed by either calculating the deviation

from the expected ratio of two maternal to one paternal genome contribution in the endosperm or spiking in material from a different accession than that used for the crosses.

Antibodies. For ChIP experiments, histone antibodies (ABs) and IgG can be used as positive and negative controls, respectively. These should bind target and nontarget regions for the mark under study and are used to test AB specificity (see an example

2n

3n

4n

6n 8n

2n

20K

50 100 150

500

100 1

4n

3n

6n 8n

a

b

c

Figure 3 | Homogenization method. (a) Microscope images of nuclei extracted using nitrogen (left) and GentleMACS (right). White arrowheads mark DAPI-stained nuclei (blue fluorescence) covered by beads (green autofluorescence). Red arrows show clusters of tissue with negative nuclei. The presence of tissue pieces in the nitrogen-based extraction explains the higher contamination level when using this extraction method. The GentleMACS dissociation was selected for the final procedure. Scale bars, 20 µm. (b) Ploidy Analyser profiles of nitrogen-extracted nuclei (left) and GentleMACS-extracted nuclei (right). 2n, 3n, 4n, 6n and 8n nuclei peaks are labeled. Broken nuclei can be shown in the nitrogen extraction plot as signal smaller than the 2n peak and the increased signal between the 2n and 4n peaks (black arrows). Importantly, the broken nuclei detected in the nitrogen-based extraction were endosperm-origin nuclei. Check supplementary Figure 2 for higher-resolution profiles obtained using GentleMACS. (c) DAPI-stained NTF-positive and NTF-negative nuclei. NTF-negative nuclei (left) show only blue fluorescence, whereas NTF-positive nuclei (right) additionally show green fluorescence from the NTF located in the nuclear envelope. Scale bars, 5 µm.

55

40

MW(kDa)

pPHE::BirA10;pPHE::NTF6

pPHE::BirA9;pPHE::NTF1

* * * *

pPHE::BirA1;pPHE::NTF6

*

*

pPH

E::B

irA10

;pP

HE

::NT

F6

pPH

E::B

irA9;

pPH

E::N

TF

6

pPH

E::B

irA9;

pPH

E::N

TF

1

pPH

E::B

irA5;

pPH

E::N

TF

6

pPH

E::B

irA1;

pPH

E::N

TF

6

pAC

T::B

irA;

pGL2

::NT

F

a

b c

Figure 2 | Generation of endosperm-specific INTACT lines with appropriate levels of NTF and BirA expression. (a) Endosperm-specific localization of the NTF fluorescence (GFP channel) in the pPHE::NTF;pPHE::BirA generated lines. The embryo nuclei (white arrowhead) do not show a fluorescence signal. Scale bars: center, 100 µm; right (applies to both right-hand panels), 40 µm. (b) Western blot (WB) with streptavidin-AB of different BirA and NTF line combinations. The arrowhead marks the NTF-specific band (42 kDa). The pACT::BirA;pGL2::NTF line2 is used as a control. (c) Binding assay (explained in Box 1) of different NTF and BirA combinations. Arrowheads mark endosperm nuclei with nuclear envelope GFP signal and stained with DAPI. Asterisks mark the M-280 streptavidin Dynabeads that show GFP channel autofluorescence. Although all the lines show a signal in the WB, not all lines show binding to the streptavidin beads (e.g., in the pPHE::BirA1;pPHE::NTF6 line). A stronger signal on WB correlates with better binding to the beads (e.g., in the pPHE::BirA10;pPHE::NTF6 and pPHE::BirA9;pPHE::NTF1 lines), and these lines were selected for performing the experiment. Scale bars, 5 µm.

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in Fig. 5a). Useful troubleshooting guidelines for the ChIP pro-cedure have been previously published and will therefore not be further detailed22–26.

Starting material. Because of the time-consuming manual pollina-tion process (Steps 1 and 2 of the procedure), the protocol has been optimized to obtain high-quality epigenome profiles with a mini-mum amount of starting material. To generate DNA methylation profiles, 100 mg of silique material is required, which is equiva-lent to ~25 siliques. For performing ChIP, the minimum amount of starting material is 200 mg for qPCR-ChIP and 400 mg for ChIP-seq experiments with three different ABs. The starting mate-rial for ChIP needs to be scaled based on the number of histone marks to be tested and also depends on the AB efficiency. Because of increasing rates of contamination, the maximum amount of starting material in one experiment is 500 mg of silique tissue. If more material is required, Steps 7–26 must be repeated and samples must be mixed at Step 27 (as shown in Supplementary Video 1).

Bioinformatic analysis for ChIP-seq. Samples are first analyzed by ignoring parental-specific information. Reads passing a quality control are mapped to the Arabidopsis (TAIR10) genome using Bowtie27 in single-end mode, allowing for up to two mismatches. Mapped reads are deduplicated and extended to the estimated average length of the genomic fragments (270 bp). Coverage is estimated and normalized to 10 million reads. H3K27me3 ChIP signals are visualized after normalization with H3 ChIP data by calculating the log2 ratio in 50-bp bins across the genome. These

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Figure 4 | Different cross-combinations tested using INT lines. (a) Endosperm fluorescence of the different crosses using the INT line as one of the parents. Before performing the experiment with new mutant combinations, it is necessary to test whether the fluorescence is maintained. Scale bars, 100 µm. (b) Pictures of binding assays using the different cross-combinations indicated in a. DAPI-stained nuclei (blue channel) covered by beads (green autofluorescence) must be present in the sample to consider the binding efficiency sufficient to perform the purification. Scale bars, 5 µm.

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Figure 5 | Endosperm-specific ChIP results. (a) ChIP followed by qPCR before library preparation. The ACTIN and PHE1 regions were used as negative and positive targets of the H3K27me3 mark, respectively. The H3 N-ter AB was used as a positive control. H3K27me3 is specifically enriched at PHE1 but not at ACTIN. Bars and whiskers represent means ± s.d., respectively. (b) ChIP-seq signals for the H3K27me3-positive region PHE1 and the H3K27me3-negative region ACTIN. The ChIP signal is represented as the log2 fold change (FC). (c) Example of parental-specific ChIP-seq and BS-seq profiles. Col × Ler and Ler × Col reciprocal crosses are shown. Total endosperm and parental-specific coverage in the different crosses is shown for the H3K27me3 mark. A leaf profile is shown for comparison. Region 1 shows tissue-specific H3K27me3, with H3K27me3 being present in the leaves but not in the endosperm. Region 2 highlights an accession-specific H3K27me3 region, with paternal-specific H3K27me3 only in Ler. Region 3 shows accession-specific CG methylation in Ler. Region 4 shows parental-specific H3K27me3 at the maternal alleles in Col and Ler. The ChIP signal is represented as log2 FC. Data shown are deposited as .fastq files in the Gene Expression Omnibus (GSE66585). bedGraphs shown are listed in supplementary table 2 and are available as supplementary Data 1–11.

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data are standardized and normalized for comparative purposes across samples with a z score transformation28 and represented in bedGraph files of 50-bp windows (Fig. 5b,c; Supplementary Data 1–11).

Parental-specific SNPs are identified by sorting reads with SNPsplit v0.2.0 (http://www.bioinformatics.babraham.ac.uk/projects/SNPsplit/). This involves mapping of all reads to an ‘N’ masked genome for all the SNP positions between the Col-0 (TAIR10) and Ler-0 (Ler_0v7) reference genomes with Bowtie 2 v2.1.0, using a seed length of 20 nt, allowing up to one mismatch per seed (-L 20 -N 1), and then by sorting the reads based on the known SNP identity. To visualize parental-specific ChIP-seq profiles, the reads are referenced to a common genomic coordi-nate system by remapping them to the TAIR10 genome, allowing for up to two mismatches per read and deduplicating, extending and normalizing them as described above.

Bioinformatic analysis for BS-seq. For each sample, the 125-bp- long pair-end reads from the Illumina BS sequence libraries should be assessed for quality control and trimmed to 100 bp by cutting 5 bp from the start and 20 bp from the end of each read. After this first trimming, each read is split into two 50-bp-long fragments. Splitting the reads and mapping them in single-end

mode improved the mapping efficiency substantially in relation to other tested options. Reads are mapped to the TAIR10 ref-erence genome using the Bismark read mapper29, allowing one mismatch per read. Duplicated reads (aligning to the same genomic position) are eliminated before calculating methylation levels. Cytosine methylation is visualized separately for CG, CHG and CHH cytosine contexts with bedGraph files representing aver-age methylation values in 50-bp windows across the genome.

Parental-specific reads are selected by mapping them as single- end to the Col-0 (TAIR10) and Ler-0 (Ler_0v7) Arabidopsis genomes, allowing zero mismatches per read. Reads that can be unequivocally assigned to one of the parents (reads aligning without mismatches to either the Col or the Ler genomes) are selected and counted. To visualize parental-specific bisulfite profiles, the reads are referenced to a common genomic coordi-nate system by remapping them to the TAIR10 genome, allowing for up to two mismatches per read.

We included information about coverage of the data repre-sented in Figure 5b,c in Supplementary Table 1. bedGraph files used to generate Figure 5b,c are listed in Supplementary Table 2 and are provided as Supplementary Data 1–11. Commented scripts used for this data analysis are listed in Supplementary Table 3 and are provided as Supplementary Data 12.

MaterialsREAGENTS

Arabidopsis seeds: The Col INT line (NASC ID N2107349, http:// arabidopsis.info/StockInfo?NASC_id=2107349) expressing pPHE1::NTF and pPHE1::BirA3 is required as either male or female parent (Fig. 4) critical Optionally, it is possible to use a male sterile mutant such as dde2 (ref. 20; INT line, NASC ID N2107350, http://arabidopsis.info/StockInfo?NASC_id=2107350) in a Col background or pistillata (pi-1)21 in a Ler background (NASC ID: NW77, http://arabidopsis. info/StockInfo?NASC_id=77) in order to omit the emasculation step. critical Use mature Arabidopsis Ler plants to collect siliques for the spike-in control (Box 3) if starting material is derived only from the Col accession.Bleach (common household bleach contains 3–6% (wt/vol) sodium hypochlorite in water, available from most supermarkets) ! caution Bleach is corrosive.Tween 20 (Merck Millipore, cat. no. 822184) ! caution Tween 20 is an irritant.Murashige and Skoog (MS) basal salt mixture (Duchefa Biochemie, cat. no. M0221) critical Optionally, any other medium or growth procedure that yields mature healthy plants can be used.Agar (Saveen Werner, cat. no. B1000)2-(N-morpholino)ethanesulfonic acid) monohydrate (MES; Duchefa Biochemie, cat. no. M1503)KOH (Sigma-Aldrich, cat. no. P5958)Liquid nitrogen (N2) ! caution Handle liquid nitrogen with care and wear appropriate personal protective equipment.Ficoll 400 (Sigma-Aldrich, cat. no. F4375)Dextran 40 (Sigma-Aldrich, cat. no. 31389)Sucrose (Duchefa Biochemie, cat. no. S0809)Tris-(hydroxymethyl) aminomethane (Tris; VWR, cat. no. 28811)MgCl2 (Sigma-Aldrich, cat. no. M8266)β-Mercaptoethanol (AppliChem, cat. no. A1108) ! caution β-Mercaptoethanol is toxic, an irritant and dangerous to the environment. Wear appropriate personal protective equipment and dispose of waste according to local and institutional regulations.Triton X-100 (Sigma-Aldrich, cat. no. X100) ! caution Triton X-100 is harmful if swallowed; wear appropriate personal protective equipment.NaCl (Merck Millipore, cat. no. 1370175000)KCl (Merck Millipore, cat. no. 104936)Na2HPO4 (Sigma-Aldrich, cat. no. 106559)

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KH2PO4 (Sigma-Aldrich, cat. no. P5655)BSA fraction V (Sigma-Aldrich, cat. no. 05482) critical Use biotin-free BSA.M-280 streptavidin Dynabeads (Invitrogen, cat. no. 112-05D)Complete Protease Inhibitor tablets (Roche, cat. no. 11873580001) ! caution Complete Protease Inhibitor tablets are toxic.Formaldehyde 37% (wt/vol) (Sigma-Aldrich, cat. no. F8775) ! caution Formaldehyde is highly toxic and volatile. It is harmful if inhaled or absorbed through the skin. Dispose of waste according to local and institutional regulations. Formaldehyde polymerizes in solution after long storage (white precipitation), which results in a reduction of the stock concentration. It is advisable to use small and fresh formaldehyde stocks.Glycine (Merck Millipore, cat. no. 104201)EDTA (Sigma-Aldrich, cat. no. 03650)SDS (Sigma-Aldrich, cat. no. L3771) ! caution Handle solid SDS with care when making solutions, as inhalation is harmful.DAPI (Invitrogen, cat. no. D9542) ! caution DAPI is both toxic and mutagenic; handle it with care.ABs: These depend on the histone modification to be investigated. The corresponding total histone must be used as a positive control and IgG as a negative control (IgG sample is not included in the sequencing, but it is necessary for ChIP-qPCR). We have successfully used the following ABs: anti-histone H3 C-ter (Upstate Millipore, cat. no. 07-690), anti-histone H3 N-ter (Sigma-Aldrich, cat. no. H9289), anti-H3K27me3 (Millipore, cat. no. 07-449), anti-H3K9me2 (Diagenode, cat. no. pAb-060-050), anti-H3K27me1 (Diagenode, cat. no. pAB-045-050) and IgG from rabbit serum (Sigma-Aldrich, cat. no. I5006) critical The AB efficiency must be tested before starting the procedure. The efficiency of different lots may vary.Dynabeads protein A (Invitrogen, cat. no. 10001D) critical Depending on the AB, use either protein A or G DynabeadsIPure Kit v2 (Diagenode, cat. no. C03010015)Nuclease-free waterMicroPlex Library Preparation Kit (Diagenode, cat. no. C05010010) or Ovation Ultralow Library System (NuGEN, cat. no. 0344 or 0330)MagJET Plant Genomic DNA Kit (Thermo Fisher Scientific, cat. no. K2761)Ovation Ultralow Methyl-Seq Multiplex System (NuGEN, cat. no. 0335)EpiTect Fast DNA Bisulfite Conversion Kit (Qiagen, cat. no. 59824)qPCR reagents (SYBR-Mix; SsoAdvanced Universal SYBR Green Supermix; Bio-Rad, cat. no. 1725270, or equivalent)

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Oligos:Tα1-2-Fw 5′-CTATGGCGAACGATCCAAAT-3′; Tα1-2-Rv 5′-GCGTCTTCCATAGCGAGAAG-3′; ACT7-Fw 5′-TGGTTTTGCTGG TGATGATG-3′; ACT7-Rv 5′-CCATGACACCAGTGTGCCTA-3′ (Sigma-Aldrich, desalt quality)

EQUIPMENTPlant growth chamberSterile plastic Petri dish, 92 × 16 mm (Sarstedt, cat. no. 82.1472.001)Sterile hoodFine forceps (Dumont, model no. 5 or equivalent)SpatulaFunnelsMortar and pestle (HandelWanger, cat. nos. 55 and 56 or equivalent) Scalpel blades, no. 22 (Feather Safety Razor, cat. no. 02.015.00.022)Binocular magnifier (Leica, model no. M60 or equivalent)50-ml Plastic tubes (Sarstedt, cat. no. 62.547.254)15-ml Plastic tubes (Sarstedt, cat. no. 62.554.001)1.5-ml Plastic tubes (Sarstedt, cat. no. 72.690.001)1-ml Micropipette tip (VWR, cat. no. 6130738)0.2-ml Nuclease-free tubes (Fisher Scientific, cat. no. 14-230-229)10-ml Serological pipette (Sarstedt, cat. no. 86.1254.001)Serological pipetting device (VWR, Accurpette or equivalent)Fine brush, no. 0 (Dekorima, 755 Marten hair or equivalent). Clean the brush by washing it with abundant distilled water for reuse.GentleMACS dissociator (Miltenyi Biotec, cat. no. 130-093-235)GentleMACS M tubes (Miltenyi Biotec, cat. no. 130-093-236)MiniMACS separation magnet (Miltenyi Biotec, cat. no. 130-042-102)Rotating mixer (Invitrogen, HulaMixer or equivalent)Miracloth (Merck Millipore, cat. no. 475855-1R)CellTrics, 30-µm cell strainer (Sysmex, cat. no. 04-0042-2316)Refrigerated centrifuge (Eppendorf, model no. 5804 R or equivalent)Mini Centrifuge (VWR, Galaxy mini centrifuge or equivalent)Refrigerated microcentrifuge (Thermo Scientific, Haraeus Fresco 17 or equivalent)Bioruptor Plus (Diagenode, cat. no. B01020001)Fluorescence microscope (Leica, model no. DMI 4000 or similar)Microscope slides and coverslipsqPCR cycler (Bio-Rad, CFX Connect Real-Time System or equivalent)Quant-IT dsDNA HS Assay Kit on the Qubit system (Invitrogen) or similarThermomixer (Eppendorf Thermomixer comfort or equivalent)Concentrator (Eppendorf Concentrator plus or equivalent)Bioanalyzer (Agilent 2100, cat. no. G2939A, or similar)Compact flow cytometer system (Sysmex, CyFlow Ploidy Analyser or equivalent)

REAGENT SETUPSeed sterilization solution Add 3 ml of bleach and 2 µl of Tween 20 to 18 ml of ddH2O. Store the solution at room temperature (20–25 °C), protected from light, for up to 1 week.1/2 MS plates Dissolve 2.3 g of MS, 0.5 g of MES and 10 g of sucrose in 800 ml of ddH2O. Adjust the pH to 5.6–5.8 using 1 M KOH. Adjust with ddH2O to a final volume of 1 liter. Add 8 g of agar and autoclave the medium for 20 min at 121 °C. Cool the medium to ~60 °C and pour it into Petri dishes in a clean sterile hood. Once solidified, store the plates at 4 °C for at least 3 months.Plant growth conditions Surface-sterilize the seeds (10 min in seed sterilization solution, and wash them three times with sterile water), stratify for 2–3 d at 4 °C and germinate them on 1/2 MS plates under long-day conditions (16 h light/8 h darkness; 21 °C). Transfer the plants to soil after 10–12 d and grow them under long-day conditions until the plants have at least 4–6 inflorescences.Sucrose, 2 M Dissolve 342.3 g of sucrose in ddH2O. Use agitation and heat to completely dissolve the sucrose. Make up the volume to 500 ml with ddH2O. Store the solution at 4 °C for up to 3 months or frozen for up to 1 year.Tris-HCl, 1 M, pH 7.4 or 8.0 Dissolve 121.14 g of Tris in ddH2O. Adjust the pH of the solution to 7.4 or 8.0 using HCl. Make up the volume to 1 liter with ddH2O, autoclave the solution and keep it at room temperature for up to 1 year.MgCl2, 1 M Dissolve 9.52 g of MgCl2 in ddH2O and make up the volume to 100 ml with ddH2O, autoclave the solution and keep it at room temperature for up to 1 year.Triton X-100, 20% (vol/vol) Add 20 ml of Triton X-100 to 80 ml of ddH2O, and use a magnetic stirrer bar to mix completely. Keep the solution at

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room temperature for up to 1 year. ! caution Wear appropriate personal protective equipment while preparing the solution.Protease inhibitor solution (25×) Dissolve one Complete Protease Inhibitor tablet in 2 ml of ddH2O. Keep the solution at −20 °C for up to 1 month.EDTA, 0.5 M, pH 8.0 Dissolve 14.61 g of EDTA in ddH2O. Use agitation and slowly add ~2 g of NaOH to the solution to adjust the pH to 8.0. Make up the volume to 100 ml with ddH2O, autoclave the solution and keep it at room temperature for up to 1 year.SDS, 20% (wt/vol) Dissolve 20 g of SDS in ddH2O. Use agitation and heat to completely dissolve the SDS. Make up the volume to 100 ml with ddH2O and keep the solution at room temperature for up to 6 months. ! caution Work in the fume hood, as solid SDS inhalation is harmful. Clean the weighing area afterward, as solid SDS crystals disperse easily.NaCl, 5 M Dissolve 146.1 g of NaCl in ddH2O. Make up the volume to 500 ml with ddH2O, autoclave the solution and keep it at room temperature for up to 1 year.Glycine, 2 M Dissolve 15.01 g of glycine in ddH2O. Make up the volume to 100 ml with ddH2O, autoclave the solution and keep it at room temperature for up to 1 year.Nuclei extraction buffer (Honda buffer) Slowly add 2.5 g of Ficoll 400 and 5 g of dextran T40 to 60 ml of ddH2O while stirring to dissolve. While stirring, add 20 ml of 2 M sucrose, 2.5 ml of 1 M Tris, pH 7.4, and 1 ml of 1 M MgCl2. Make up the volume to 100 ml with ddH2O. Filter-sterilize the solution and make 10-ml aliquots to be stored at 4 °C for up to 6 months. Just before use, add 250 µl of 20% (vol/vol) Triton X-100 and 7 µl of β-mercaptoethanol. 10 ml of solution is required per extraction.PBS (10×) Dissolve 80 g of NaCl, 2 g of KCl, 14.4 g of Na2HPO4 and 2.4 g of KH2PO4 in ddH2O. Adjust the pH to 7.4 with KOH. Make up the volume to 1 liter with ddH2O, autoclave the solution and keep it at room temperature for up to 1 year.PBS (1×) Dilute PBS (10×) in a 1:9 ratio with ddH2O. Keep the solution at room temperature for up to 1 year.PBSB buffer Dissolve 50 mg of BSA in 50 ml of PBS. Make the buffer fresh and keep it on ice before use. 20 ml of solution is required per extraction.PBSBt buffer Add 60 µl of 20% (vol/vol) Triton X-100 to 12 ml of PBSB buffer. Make it fresh and keep it on ice before use. 12 ml of solution is required per extraction.Nuclei lysis buffer Add 25 µl of 1 M Tris-HCl, pH 8.0, 10 µl of 0.5 M EDTA, 50 µl of 10% (wt/vol) SDS and 20 µl of Protease Inhibitor solution (25×) to 395 µl of ddH2O. Make the buffer fresh and keep it at room temperature until use.ChIP dilution buffer Add 550 µl of 20% (vol/vol) Triton X-100, 24 µl of 0.5 M EDTA, 167 µl of 1 M Tris-HCl, pH 8.0, 334 µl of 5 M NaCl and 400 µl of Protease Inhibitor solution (25×) to 8.53 ml of ddH2O. Make the buffer fresh and keep it on ice until use. Keep the remaining buffer at 4 °C until the next day to equilibrate the magnetic Dynabeads (Step 27B(xi)).Low-salt ash buffer Add 50 µl of 10% (wt/vol) SDS, 250 µl of 20% (vol/vol) Triton X-100, 20 µl of 0.5 M EDTA, 100 µl of 1 M Tris-HCl, pH 8.0, and 150 µl of 5 M NaCl to 4.43 ml of ddH2O. Keep the buffer on ice until use.High-salt wash buffer Add 50 µl of 10% (wt/vol) SDS, 250 µl of 20% (vol/vol) Triton X-100, 20 µl of 0.5 M EDTA, 100 µl of 1 M Tris-HCl, pH 8.0, and 500 µl of 5 M NaCl to 4.08 ml of ddH2O. Keep the buffer on ice until use.TE buffer To make Tris-EDTA (TE) buffer, add 50 µl of 1 M Tris-HCl, pH 8.0, and 10 µl of 0.5 M EDTA to 4.94 ml of ddH2O. Keep the buffer on ice until use.Col and Ler DNA Purify DNA from Arabidopsis Col and Ler accessions for preparing the Col:Ler DNA reference samples (Box 3). The required Col:Ler ratios needed are 1:1 (ref1:1), 1:0 (ref1:0) and 0:1 (ref0:1). critical Correctly quantify high-quality DNA from Col and Ler accessions to prepare the reference samples. The concentration of DNA in the reference samples must be in the same range as that of the sample to be analyzed.DAPI solution (1.0 mg/ml) Dissolve 0.5 mg in 500 µl of ddH2O. DAPI has poor solubility in water; sonicate if necessary to dissolve. Keep the solution stored in the dark at 4 °C for up to 3 weeks or at −20 °C for 6 months. ! caution DAPI is both toxic and mutagenic; wear appropriate personal equipment.

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proceDuretissue preparation and collection ● tiMinG 6 d critical Use flowering plants from Arabidopsis Col and Ler accessions for performing the crosses. At least one of the parents must be an INT line (in a Col background; Fig. 4). critical Check the endosperm fluorescence and perform a binding assay (Box 1) before proceeding with the purification if the INT line will be crossed with a line different from the ones shown in Figure 4. critical Steps 3 and 4 of this section can be followed using supplementary Video 1.1| Take the female parent plant (pistil donor) and remove the siliques and opened flowers. Under a binocular magnifier, remove the stamens of flower buds that have not yet opened (buds that have not yet fully developed anthers). Repeat the emasculation for other buds in the inflorescence (usually 3–5 buds per inflorescence). Place the emasculated plants in a growth chamber for 2 d. critical step To prevent the pistils from drying out, avoid direct air flow to the emasculated flowers. critical step As an alternative, use pollination-deficient or male sterile mutant plants to avoid emasculation.

2| Hold the emasculated flowers under the binocular magnifier. Using forceps, take an open flower from the plant designated as the male parent and tap the anthers onto the stigma to cover it with pollen grains. Repeat for all stigmata. Put the plants back into the growth chamber for 4 d. critical step The efficiency of the pollination should be as high as possible. Mature pistils have strongly papillated stigmata that must be covered with pollen for an efficient pollination. To prevent the pistils from drying out, avoid direct air flow to the pollinated flowers. critical step Alternatively, if the experiment can be done with self-fertilized INT plants, skip Steps 1 and 2. To ensure that only siliques at 3–4 DAP will be harvested, remove all the siliques and pollinated flowers at a defined time point, and 4 d later, harvest the older siliques (2–4 for each inflorescence branch), which correspond to 3–4 DAP. Perform the same procedure for collecting Ler material when the spike-in is needed.? trouBlesHootinG

3| Collect the siliques at 4 DAP. Count and weigh the siliques and freeze a maximum of 500 mg per sample. Wrap the material in aluminum foil. 100 mg corresponds to ~35–40 siliques.? trouBlesHootinG

4| Flash-freeze the siliques in liquid N2.! caution Handle liquid N2 with care. pause point The sample can be stored at −80 °C for up to 1 year.

nuclei extraction ● tiMinG 30 min critical Perform all steps on ice and/or in the cold room. Precool all buffers and plastic tubes on ice and cool down the centrifuges. Proceed with a maximum of two samples in parallel (in case more than 500 mg of starting material is required, perform all procedures in parallel and pool the samples in Step 27B(vii)) critical The minimum amount of plant material required is 100 mg of siliques for BS-seq experiments, 200 mg of siliques for ChIP-qPCR experiments and 500 mg of siliques for ChIP-seq (the amount of material required for ChIP increases with increasing numbers of ABs used). When siliques derived from Col × Col crosses are used, 20–30% (wt/wt) of Ler siliques should to be added to the sample (spike-in) to calculate purity. critical This section can be followed using supplementary Video 1, which shows the processing of two samples in parallel.5| Add 5 ml of nuclei extraction buffer to a GentleMACS M tube and keep it on ice.! caution Nuclei extraction buffer contains β-mercaptoethanol, so work under a fume hood.

6| Take the required number of samples from the −80 °C freezer and keep them in liquid N2.! caution Handle liquid N2 with care.

7| Take the sample from the liquid N2 and crush the siliques before removing them from the aluminum foil by pressing with a cooled spatula several times. Put the broken siliques in the GentleMACS tube with the nuclei extraction buffer and briefly shake the solution to prevent buffer freezing. critical step This step should be done as quickly as possible to avoid thawing of the siliques and to prevent them from getting stuck to the envelope. Be aware that nonbroken siliques can explode when N2 evaporates from the interior of the silique. critical step When more than 500 mg of siliques are required to be processed, handle samples as duplicates until Step 26.

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8| Insert the GentleMACS tube cap into the GentleMACS Dissociator and run the following programs:

‘m_brain_03.01’ three times; ‘h_tumor_03.01’, three times; add 2 ml of nuclei extraction buffer; ‘h_tumor_03.01’; ‘h_tumor_02.01’; ‘h_tumor_03.01’; ‘m_brain_03.01’, two times.

critical step After running each of the first three programs, it is necessary to open the tube and remove the tissue that clogs the rotor of the GentleMACS M tube. Invert the tube between programs to allow pieces of siliques that remain on the tube walls to fall down to the rotor.

9| Spin the GentleMACS tube at 400g for a few seconds at 4 °C to collect the homogenate at the bottom. Exchange the GentleMACS tube lid with a lid from a 50-ml tube and let the homogenate slowly rotate at 10 r.p.m. for 15 min at 4 °C.

10| While the tubes are rotating, pipette the M-280 streptavidin Dynabeads (each sample requires 36 µl) into a 1.5-ml tube, and then add 200 µl of PBSB buffer and resuspend the beads by pipetting up and down. Place the tube in the magnetic rack to capture the beads, and discard the supernatant. Repeat the wash once. Resuspend the beads in 200 µl of PBSB buffer and leave them preblocking under rotation at 10 r.p.m. at 4 °C for at least 30 min before use.

11| Perform a serial filtration of the homogenate (Step 9). First, decant the homogenate into a funnel placed in a 50-ml tube with a single-layer of Miracloth. Add 1 ml of nuclei extraction buffer to the GentleMACS tube to collect the remaining homogenate. Then, perform a second filtration into a 15-ml tube using a double-layer of Miracloth. critical step Wet the Miracloth with nuclei extraction buffer before filtrations, and press the Miracloth using a spatula after filtration to collect the maximum volume of homogenate.

12| Perform a final filtration using a CellTrics strainer (30-µm size selection pore) into a 15-ml tube. Add to the filter the remains of homogenate left in the tubes used in the different filtration steps. Cut the 1-ml tip before pipetting.

13| Centrifuge the homogenate at 1,500g for 6 min at 4 °C.

14| Remove the supernatant using a serological pipette. Leave some solution, <1 ml.

15| Remove the remaining supernatant using a micropipette. critical step The pellet should be small and loose. Leave some solution to avoid pipetting away nuclei.

16| Add 1,500 µl of PBSB buffer and resuspend the pellet by carefully brushing with the tip of a fine brush (also see supplementary Video 1).

17| Split the resuspended extracted nuclei over two 1.5-ml tubes (750 µl each). When pipetting, use a cut 1-ml pipette tip. critical step We recommend saving 20–50 µl as a nonpurified control (NPC) sample that can be used in the qPCR purity assessment to calculate the spike-in (S) value in the NPC sample (Snpc) (Box 3). The Snpc value can also be estimated using the weight of the Ler spike-in (Box 3). In addition, as an option for spike-in experiments, use an aliquot to calculate the number of positive and negative fluorescent nuclei. The ratio of the two values will be used to calculate the Enpc value (ratio of INTACT positive and negative nuclei), which is required for the final purity calculations (Box 3). The Enpc value can also be calculated using a Ploidy Analyser (compact flow cytometer system; supplementary Fig. 2).

purification of labeled nuclei ● tiMinG 90 min critical Do all the pipetting involving nuclei with cut tips. All the steps are performed on ice and/or in a cold room. Precool the required buffers on ice and precool the centrifuges. critical This section can be followed from Steps 18–27 (followed by Step 27B(i–viii)) in supplementary Video 1, showing the processing of two samples in parallel.18| Place the tubes containing preblocked M-280 streptavidin Dynabeads (Step 10) in the magnetic rack. Remove the supernatant and resuspend in 36 µl of PBSB buffer.

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19| Add 18 µl of preblocked streptavidin beads to both of the nuclei extraction tubes (from Step 17) and incubate them under rotation for 30 min at 4 °C.

20| While the tubes in Step 19 are being incubated, preblock several 1-ml tips with PBSB buffer. To do this, take 1 ml of PBSB buffer into the 1-ml tips and leave the solution inside. One tip per purification is required. Place the tips in a cold room for later use (Step 24).

21| Using a mini centrifuge, spin the two tubes of incubated extracted nuclei (Step 19) for a few seconds at room temperature to remove the volume from the lid, and then place them in the magnetic rack. Discard the supernatant. Resuspend the beads in 500 µl of PBSBt buffer by inverting the tube several times.

22| Using a mini centrifuge, spin the tubes for few seconds at room temperature to remove the volume from the lid. Combine the contents of the two tubes of PBSBt-buffer-resuspended beads in a 15-ml tube with 11 ml of PBSBt buffer. Use a cut tip and do it slowly.

23| Incubate under rotation for 15 min.

24| Prepare the column-like separation system: add to the preblocked tip (Step 19) a system that allows you to control the flux of the liquid4. Insert the 1-ml tip into the miniMACS magnet. Fill the tip with PBSB buffer. Avoid bubbles in the system. critical step Alternatively, use a 15-ml magnetic rack30.

25| Slowly pipette the beads incubated with PBSBt buffer (Step 22) into a 10-ml serological pipette, and insert the filled pipette on top of the tip placed in the magnet. Avoid air bubbles.

26| Allow the liquid to flow through the magnet at a speed of ~2 ml/min. All the solution should pass through and the tip should be completely empty at the end.

27| Remove the tip from the magnet using the micropipette, cut the tip, and pipette up and down in the appropriate buffer. Depending on the downstream application, follow the different steps for DNA methylation analysis by BS-seq (option A) or histone modification analysis by ChIP (option B).(a) Bs-seq ● tiMinG 12 h critical step Read the instruction manuals for the MagJET Plant Genomic DNA kit and the Ovation Ultralow Methyl-Seq Library Kit carefully before following our adapted steps. (i) Resuspend the captured beads in 350 µl of lysis buffer A from the MagJET Plant Genomic DNA Kit. (ii) Remove the beads using the magnetic rack. (iii) Follow the MagJET Plant Genomic DNA Kit supplier protocol (instructions for manual genomic DNA purification)

and perform the final DNA elution in 80 µl of elution buffer from the kit. (iv) Transfer the purified DNA to a 1.5-ml tube.

pause point The DNA can be stored at 4 °C for 24 h or −80 °C for up to 6 months. (v) Assess purity using qPCR (Box 3).

? trouBlesHootinG (vi) Fragment the DNA in the Bioruptor Plus using the high-power setting for 30 cycles (30 s ON, 30 s OFF).

critical step Sonication may cause the solution to cling to the walls of the tube; spin down the sample, if required, during the sonication cycles.

(vii) Quantify the samples using a Quant-IT dsDNA HS Assay Kit on the Qubit system. ? trouBlesHootinG

(viii) Take 10 ng of sample for library preparation (10 ng is the minimum recommended amount of starting material). The DNA purification step is not required when using the Ovation Ultralow Methyl-Seq Library kit, and it is possible to proceed directly with the end repair step. If the DNA is too diluted, concentrate the sample in a concentrator until the 13-µl starting volume required for the library protocol is obtained.

(ix) Follow the supplier’s manual to generate the library. For bisulfite conversion, use the EpiTect Fast Bisulfite Conversion Kit. ? trouBlesHootinG

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(x) Use barcodes in case you want to pool the libraries for sequencing. 4-plex libraries have been successfully sequenced (HiSeq Illumina). critical step Consider that the Methyl-Seq libraries require the use of the custom read 1 sequencing primer, MetSeq Primer 1, and that this primer must be mixed together with the standard read 1 sequencing primer for the other Illumina standard libraries or a PhiX library. When using other standard libraries, make sure that the barcodes are at a suitable distance from those of the Methyl-Seq libraries. It is also recommended to reduce the cluster density to 75% of your normal density.

(B) chip ● tiMinG 3 d critical step Precool the sonicator and centrifuges. Perform all steps on ice and/or in the cold room unless indicated. Multiplex libraries can be generated. 12/16-plex for ChIP-seq has been performed successfully. Step 27B(i–ix) can be followed using supplementary Video 1. (i) Slowly resuspend the beads in 500 µl of PBSB buffer.

critical step Do the pipetting with a cut tip. (ii) Add 13.5 µl of 37% (wt/vol) formaldehyde (final concentration 1% (wt/vol)) and incubate on ice for 8 min.

Invert the tube two to three times during incubation. ! caution This mixture is toxic; work in a fume hood.

(iii) Soft-spin for few seconds at room temperature using a mini centrifuge to collect the liquid caught in the tube cap and add 32.25 µl of 2 M glycine (to a final concentration of 125 mM) and incubate for 5 min. Invert the tube two to three times during incubation.

(iv) Spin for few seconds at room temperature using a mini centrifuge to collect the liquid caught in the tube cap. Put the tube in the magnetic rack for 1 min (or until the beads are trapped), and remove the supernatant.

(v) Add 100 µl of nuclei lysis buffer. Resuspend the beads by pipetting up and down, avoiding foam formation. critical step If samples must be pooled, resuspend them in 100-µl total volume.

(vi) Sonicate the chromatin solution in the Bioruptor Plus using the high-power setting for nine cycles (20 s ON, 45 s OFF). critical step Check after half of the cycles have been performed to determine whether spinning down of the sample is required. Place extra tubes in the empty spaces of the sonicator to allow homogeneous sonication. critical step If you are adapting the protocol to a tissue other than the endosperm or if you use a sonicator different from the Bioruptor Plus, make a prior test of sonication efficiency. Sonication should yield DNA fragments ranging from 0.3 to 1.5 kb in size. Sonication efficiency should be tested using nuclei from the same tissue isolated with the same extraction method and in comparable amounts. To visualize the fragment size on an agarose gel, 30–50 ng of DNA is required (de-cross-linked and RNAse-treated) before and after sonication. Pooling of samples that have been sonicated in parallel may be required to reach detectable amounts of DNA. ? trouBlesHootinG

(vii) Add 900 µl of ChIP dilution buffer to dilute the SDS concentration from 1 to 0.1% (wt/vol). (viii) Centrifuge for 5 min at >8,000g at 4 °C and collect the supernatant. (ix) Split the chromatin solution into the necessary number of tubes (one for each IP; use IgG as a negative control and

total H3 as a positive control) and keep aside 20 µl of chromatin as input (keep it at −20 °C). Add the appropriate amount of ABs to the tubes. critical step The amount of AB needs to be determined (typically 1 µg of AB per 100 µl of chromatin solution).

(x) Incubate the tubes overnight at 4 °C with rotation. (xi) Equilibrate the magnetic Dynabeads by washing them two times with ChIP dilution buffer, and resuspend the beads

in the same buffer. Place the required volume of Dynabeads (typically the amount of beads required is 1/10th the volume of the chromatin) into a 1.5-ml tube, add 200 µl of PBSB buffer, and resuspend the beads by pipetting up and down. Place the tube in the magnetic rack to capture the beads, and discard the supernatant. Repeat the wash. Resuspend the beads in the original volume with ChIP dilution buffer.

(xii) Add the proper volume of equilibrated Dynabeads to each ChIP aliquot. Rotate at 4 °C for 90 min. (xiii) Spin for a few seconds at room temperature using a mini centrifuge to collect liquid caught in the tube cap,

and capture the beads with a magnetic rack. Discard the supernatant. (xiv) Remove the tubes from the magnet. Add the same volume of low-salt wash buffer as that of the chromatin

used for the IP. Resuspend the beads by inverting the tube, spin for a few seconds at room temperature using a mini centrifuge to collect liquid caught in the tube cap and capture the beads with the magnetic rack. Discard the supernatant.

(xv) Add the same volume of low-salt wash buffer, resuspend the beads by inverting the tube and rotate for 5 min at 4 °C. (xvi) Spin for few seconds at room temperature using a mini centrifuge to collect liquid caught in the tube cap, and

capture the beads with a magnetic rack. Discard the supernatant.

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(xvii) Remove the tubes from the magnet; add the same volume of high-salt wash buffer as that of the chromatin used for ChIP. Resuspend the beads by inverting the tube, spin for a few seconds at room temperature using a mini centrifuge to collect liquid caught in the tube cap and capture the beads with the magnetic rack. Discard the supernatant.

(xviii) Add the same volume of high-salt wash buffer, resuspend the beads by inversion and rotate at 4 °C for 5 min. (xix) Spin the mixture for a few seconds at room temperature using a mini centrifuge to collect the liquid caught

in the tube cap, and capture beads with a magnetic rack. Discard the supernatant. (xx) Remove the tubes from the magnet; add the same volume of TE buffer as that of the chromatin used for ChIP.

Resuspend the beads, spin for a few seconds at room temperature using a mini centrifuge to collect the liquid caught in the tube cap and capture the beads on the magnetic rack. Discard the supernatant.

(xxi) Remove the input sample (Step (ix)) from the refrigerator. (xxii) Add 100 µl of buffer A+B from the Ipure Kit to each IP tube (from Step (xx)) and 180 µl to the 20-µl input

(from Step (xxi)). Follow the IPure Kit protocol until the elution step. critical step Adjust the protocol to the volume of the input sample. critical step The recovery of DNA with the Ipure Kit is generally high and the quality is sufficient to continue directly with the library preparation. However, if desired, phenol/chloroform DNA extraction with additional XP bead washing can be done before library preparation. pause point The solution with the elution buffer can be stored at 4 °C for 24 h or −20 °C for 2–3 d, but remove the magnetic beads before freezing.

(xxiii) Place the tubes for de-cross-linking at 65 °C for least 6 h or overnight in a Thermomixer with continuous shaking. (xxiv) Allow the tubes to reach room temperature and soft-spin for a few seconds at room temperature using a mini

centrifuge to collect the liquid caught in the tube cap. (xxv) Capture the beads with a magnetic rack, and collect the supernatant.

critical step After a long incubation at 65 °C, the tubes may not close tightly, especially when organic compounds are used. Transfer the solution to new 1.5-ml tubes before continuing. pause point DNA can be stored at 4 °C for 24 h or at −20 °C for a week.

(xxvi) Continue with the IPure Supplier protocol. (xxvii) Elute twice by incubating for 15 min on a shaker at room temperature. Use 60 µl of water for the first elution

and 20 µl of elution buffer (buffer C from the kit) for the second. Combine the two eluates. critical step Allow the beads to become totally dry before the first elution. pause point DNA can be stored at 4 °C for 24 h or at −20 °C for a week.

(xxviii) Before library preparation, test by qPCR to determine whether the IP was successful. Samples can be diluted for the qPCR control. Positive and negative AB controls (total H3 and IgG), and positive and negative target regions (for qPCR) are required. Assess the purity as indicated in Box 3. ? trouBlesHootinG

(xxix) Quantify the samples using a Quant-IT dsDNA HS Assay Kit on the Qubit system. Do not use more than 8 µl of the sample for quantification. critical step If you need to use >8 µl for quantification, the amount of DNA in the sample is <1 ng. If quantification is not possible, concentrate the whole sample and use the MicroPlex Library Preparation Kit (<1 ng required). Nevertheless, total H3 and input samples should amount to 1 ng; otherwise, the protocol has probably failed. ? trouBlesHootinG pause point DNA can be stored at −80 °C for up to 6 months.

(xxx) Sonicate the samples in the Bioruptor Plus using the high-power setting for 25 cycles (30 s ON, 30 s OFF). critical step This step can be omitted if longer fragment libraries are required.

(xxxi) Library preparation. Pipette the volume that corresponds to 1 ng. Concentrate the samples to the desired starting volume in a concentrator. pause point DNA can be stored at 4 °C overnight before starting library preparation the next day.

(xxxii) Use the appropriate library kit. Use the Microplex Library Preparation Kit (when more than 16-plex libraries are required or <1 ng is recovered) or the Ovation Ultralow Library System (from 1–16-plex libraries; at least 1 ng of starting material is required). ? trouBlesHootinG

? trouBlesHootinGTroubleshooting advice can be found in table 1.

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taBle 1 | Troubleshooting table.

step problem possible reason solution

2 Insufficient pollen Pollen donor plants were not correctly watered

Add water the night before

It is late afternoon The maximum pollen release is in the morning; pollinate before the afternoon

3 Siliques are short; few seeds are obtained

The pistil used for pollination became dry, and was not mature enough or was too old

The mature pistil has a papillated stigma. Premature stages have small papillae, and when pistils are too old the papillae appear thin. Choose the correct stage. Papillae are absent if the pistil is dried out. Protect the emasculated flowers from direct air flow

Too few pollen grains on the pistil Cover the pistil with pollen grains

27A(v), 27B(xxviii) and Box 3

Contaminated extracted nuclei

Too much starting material For >500 mg of starting material, perform the procedure in parallel and pool the samples in Step 27B(vii)

Too much debris after homogenization

Add an extra filtration step, homogenize for a shorter time and change the homogenization protocol (Box 2)

Problems with qPCR assay Adjust reference DNA concentrations

27A(ix) and 27B(xxxii)

Insufficient library amplification

Too few cycles It is recommended to perform 11–14 cycles. It is useful to take 10 µl of sample from the last amplification step and purify the DNA with AMPure beads. If the yield is not sufficient, add two to three additional cycles to the remaining reaction mixture

27A(vii) Insufficient DNA for bisulfite-seq (<10 ng)

Too little starting material Increase the amount of starting material. In the case that >500 mg is required, pool samples from parallel extractions

Low pollination efficiency and few seeds per silique

Increase the pollination efficiency or increase the number of siliques

27B(vi) DNA fragments are too large

Inefficient sonication program Add more sonication cycles

Drops formed on the wall of the tube wall during sonication

Spin down the tube during sonication

27B(xxviii) High background (IgG levels)

Over-cross-linking Check formaldehyde concentration

Chromatin not correctly shared (<1.5 kb)

Check the sonication efficiency before the procedureAvoid drops on the tube wall during sonication

Homogenization was too strong Change the extraction method (Box 2)

27B(xxix) Low yield of ChIP DNA

Too little starting material Increase the amount of starting material. In the case that >500 mg is required, pool samples from parallel extractions

Poor efficiency of the AB Increase the amount of AB or change it. Test each new AB batch before starting the procedure

Too much nonspecific binding A preclearing step can be included if high background is detected in the PCR analysis. The use of Dynabeads reduces the background and allows avoiding preclearing

Box 1, step 13

Too few nuclei in the extract

Too little starting material Do not use <100 mg

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● tiMinGStep 1, flower emasculation: 2–4 h, 2 d of pistil maturationStep 2, flower pollination: 2–4 h, 4 d of silique developmentSteps 3 and 4, silique collection: 15–20 minSteps 5–17, nuclei extraction: 30 minSteps 18–26, purification of labeled nuclei: 90 minstep 27a, Bs-seqStep (i–iv), gDNA purification: 60 minStep (v), assessment of purity by qPCR (Box 3): 2 hStep (vi–x), library preparation: 5–9 hstep 27B, chipStep (i–vi), cross-linking and sonication: 25 minStep (vii–x), chromatin–AB incubation: 15 min and overnightStep (xi–xx), chromatin–IP recovery and washes: 2 hStep (xxi–xxiii), de-cross-linking: 6 h or overnightStep (xxiv–xxvii), DNA purification: 100 minStep (xxviii–xxix), qPCR and DNA quantification: 2 hStep (xxx–xxxii), library preparation: 3–5 hBox 1, binding assay: 2 hBox 2, nuclei extraction methods: variableBox 3, purity assay qPCR: 2 h

anticipateD resultsEndosperm nuclei purification from 100 mg of siliques using the INTACT system yields typically 20 ng of genomic DNA, but varies from 5–50 ng depending on the input material. This amount of DNA is sufficient for performing BS-seq experiments. For ChIP-seq experiments, ~0.8–1 g of starting material is required to test three histone modifications (H3K27me3, H3K9me2 and H3K27me1) and histone H3 by ChIP, yielding ~1 ng of DNA. As the amount of DNA after IP depends on the AB efficiency and the abundance of the modification, different volumes of chromatin can be used for each AB, based on empirical results. Following the purification protocol, the calculated contamination is expected to be <10%, never exceeding 15%. The number of reads obtained after sequencing allows the user to distinguish between maternal and paternal sequences and to generate parental-specific epigenome profiles (Fig. 5, quality details in supplementary table 1).

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

acknoWleDGMents This research was supported by a European Research Council Starting Independent Researcher grant (to C.K.), a grant from the Swedish Science Foundation (to C.K.), a grant from the Knut and Alice Wallenberg Foundation (to C.K.) and a grant from the Royal Physiographic Society in Lund (to J.M.-R.). We thank A. Ortiz Herrera for recording and editing the video.

autHor contriButions J.M.-R. and C.K. designed the research; J.M.-R. performed the experiments; J.M.-R., J.S.-G. and C.K. analyzed the data; L.H. developed the formula; and J.M.-R. and C.K. wrote the manuscript.

coMpetinG Financial interests The authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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