May 04, 2026

FANS-free nuclei isolation from green plant tissues for single nucleus RNA-seq and ATAC-seq

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4pne8lo5/v1
  • Lee J. Conneely1,2,3
  • 1La Trobe Institute for Sustainable Agriculture and Food, La Trobe University, AgriBio Building, Bundoora, VIC, 3086, Australia;
  • 2Australian Research Council Research Hub for Medicinal Agriculture, La Trobe University, AgriBio Building, Bundoora, VIC, 3086, Australia;
  • 3Australian Research Council Centre of Excellence in Plants for Space, La Trobe University, Bundoora, VIC, Australia
  • single cell
Lee Conneely
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl4pne8lo5/v1
Protocol Citation: Lee J. Conneely 2026. FANS-free nuclei isolation from green plant tissues for single nucleus RNA-seq and ATAC-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4pne8lo5/v1
Manuscript citation:

License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 24, 2026
Last Modified: May 04, 2026
Protocol  Integer ID: 315665
Keywords: free nuclei isolation from green plant tissue, optimized nuclei isolation, 10x genomics single cell multiome atac, single nucleus rna, nuclei isolation protocol, single cell multiome atac, nuclei isolation, free nuclei isolation, lee conneely from the nuclei isolation protocol, nucleus multiome profiling, green plant tissue, separation of nuclei, chloroplast, nuclei preparation, gene expression sequencing, rosaceae species, gene expression workflow, 10x genomic, leaves from solanaceae, solanaceae, nuclei
Funders Acknowledgements:
Australian Research Council Industrial Transformation Hub in Medicinal Agriculture
Grant ID: IH180100006
Abstract
This protocol describes an optimized nuclei isolation workflow suitable for two nuclei preparations and compatible with the 10x Genomics Single Cell Multiome ATAC + Gene Expression workflow. The method was modified by Lee Conneely from the nuclei isolation protocol of Valkonen et al. (2013), originally developed for leaves from Solanaceae and Rosaceae species, with additional buffer and wash-step modifications adapted from the 10x Genomics nuclei isolation protocol for Single Cell Multiome ATAC + Gene Expression Sequencing (CG000365, Rev C).

In the original Valkonen protocol, an extended centrifugation step is used to improve separation of nuclei from chloroplasts. Here, this centrifugation time was reduced to shorten sample handling and limit potential transcriptional, chromatin, or stress-associated artefacts introduced during nuclei isolation. As a result, nuclei and chloroplasts may initially co-isolate. Plastid contamination is subsequently reduced during the lysis and wash steps by optimizing Triton X-100 concentration, enabling recovery of intact, permeabilised, nuclei suitable for downstream single-nucleus multiome profiling.
Guidelines
  1. This protocol was optimised for isolating nuclei from fresh green Cannabis sativa floral tissue for downstream 10x Genomics single-nucleus Multiome workflows. Plant species and tissue types differ in nuclei size, nuclei fragility, plastid abundance and tissue composition; therefore, detergent concentration, filter pore size and handling steps may require empirical optimisation when adapting this protocol to other species.
  2. For Cannabis sativa, nuclei were generally approximately 3–10 µm. In tomato, some nuclei were larger than 10 µm, and replacing the 10 µm mini PluriStrainer with a 20 µm strainer improved nuclei recovery. Filter size should therefore be selected based on the expected nuclei size and the amount of debris or clumping in the preparation.
  3. Plastid contamination may be visible as a green pellet or green band during the Percoll‱ cleanup. When this protocol was adapted for tomato, increasing Triton X-100 from 0.2% to 0.5% improved plastid removal. However, this should be optimised cautiously, as higher detergent concentrations can damage nuclei and tolerance may differ between species.
  4. During Percoll‱ cleanup, high-quality intact nuclei may co-localise with chloroplasts/plastids at the interface between the upper aqueous layer and the 30% Percoll‱ layer. A darker green plastid-rich band may form lower in the gradient between the 30% and 70% Percoll‱ layers and should be avoided. If applying this protocol to other species it is worthwhile examining each layer by fluorescence microscopy with a suitable DNA binding dye, to see where the high-quality nuclei settle.
  5. Nuclei do not remain evenly suspended and will gradually settle to the bottom of the tube. Gently resuspend nuclei immediately before counting and again before downstream dilution, tagmentation or 10x loading.
Materials

ABCD
Nuclei Wash Buffer StockFinal7.5 mL (3750 µL /sample)
Tris pH 6.91 M10 mM75 µL
NaCl5 M10 mM15 µL
MgCl21 M3 mM22.5 µL
CaCl21 M5 mM37.5 µL
BSA10%1 %750 µL
Tween-2010%0.1%75 µL
DTT1 M1 mM7.5 µL
RNase Inhibitor40 U/µL1 U/µL187.5 µL
Nuclease Free Water--6,330 µL
Nuclei wash buffer recipe (NWB). Fixed volume recipe D is sufficient for two samples using the associated protocol.


ABCD
Nuclei Lysis BufferStockFinal2 mL (1 mL /sample)
Tris pH 6.91 M10 mM20 µL
NaCl5 M10 mM4 µL
MgCl21 M3 mM6 µL
CaCl21 M5 mM10 µL
BSA10%1 %200 µL
Tween-2010%0.1%20 µL
Triton X-10010%0.2%*40 µL*
DTT1 M1 mM2 µL
RNase Inhibitor40 U/µL1 U/µL50 µL
Nuclease Free Water--1648 µL
Nuclei lysis buffer recipe. *Triton X-100 final concentration may need to be optimised for nuclei from other species



ABCD
Diluted Nuclei BufferStockFinal600 µL (300 µL /sample)
10X GenomicsNuclei Buffer (20X)20X1X30 µL
DTT1 M1 mM0.6 µL
RNase Inhibitor40 U/µL1 U/µL15 µL
Nuclease Free Water--554.4 µL
Diluted 10X Genomics nuclei buffer (1X)


ABCD
30% Percoll™ StockFinal400 µL (200 µL /sample)
PercollTM100%30%120 µL
Wash Buffer1X70%270 µL
RNase Inhibitor40 U/µL1 U/µL10 µL
30% Percoll Solution. New wash buffer should be made for this recipe please refer to nuclei wash buffer recipe




ABCD
70% Percoll™ StockFinal400 µL (200 µL /sample)
PercollTM100%70%280 µL
Wash Buffer1X30%112.5 µL
RNase Inhibitor40 U/µL0.75 U/µL7.5 µL
70% Percoll Solution. New wash buffer should be made for this recipe please refer to nuclei wash buffer recipe

  • Percoll (Sigma-Aldrich P4937)
  • RNase protector inhibitor (Roche 03335399001)
  • Tris-HCl
  • NaCl
  • MgCl2
  • CaCl2
  • BSA (Sigma-Aldrich A9647)
  • Tween-20 (Bio Rad 1610781)
  • DTT (Thermo Fisher Scientific A39255)
  • Triton X-100 (Sigma-Aldrich X-100)
  • 10x Genomics 20X concentrated Nuclei buffer (10x Genomics PN-2000207)
  • Feather Blades – Hi-Stainless Platinum Coated Double Edge Blades
  • 20 μm mesh size mini pluristrainers (pluriSelect 43-10020-50)
  • LoBind Eppendorf tubes 2mL (Eppendorf 022431048)
  • 10 μm mesh size mini pluristrainer (pluriSelect 43-10010-50)
  • Hoescht 33342 (Thermo FischerScientific H3570)
  • haemocytometer


Troubleshooting
Problem
Green pellet persists
Solution
Increase Triton X-100 cautiously in nuclei lysis buffer or extend lysis time. Alternatvely, increase Triton X-100 concentration in first post-lysis wash step.
Problem
Low nuclei recovery
Solution
consider using a larger filter pore size.
Problem
Large clumps dominate
Solution
re-filter through 10 µm or 20 µm mesh. Reduce Triton X-100 concentration in lysis buffer or reduce lysis time.
Problem
Poor nuclei integrity
Solution
reduce chopping time, pipetting force, detergent concentration or total handling
Safety warnings
  1. Do not omit or substitute Protector RNase Inhibitor when preparing nuclei for downstream 10x Genomics single-nucleus Multiome or other RNA-based single-nucleus workflows. Using the wrong RNase inhibitor, or omitting RNase inhibitor, can severely compromise RNA recovery.
  2. Prepare all buffers fresh on the day of the experiment. Add Protector RNase Inhibitor immediately before use. Prepare 30% and 70% Percoll‱ solutions using Wash Buffer without RNase inhibitor, as the inhibitor concentration is already accounted for in the Percoll‱ recipes.
  3. Do not over-chop the tissue. Excessive chopping, rapid chopping or prolonged chopping can damage nuclei integrity. The goal is to generate a slurry-like consistency while preserving intact nuclei.
  4. Avoid vigorous pipetting, vortexing or rapid resuspension at all stages. Nuclei are fragile and easily damaged by mechanical shearing.
  5. Do not stain the nuclei aliquot intended for downstream tagmentation or 10x loading. DNA-binding dyes should only be added to the separate aliquot used for counting and microscopy.
  6. Estimate nuclei concentration and quality as quickly as possible, then proceed immediately to downstream nuclei dilution and tagmentation. Long delays can reduce nuclei quality and may affect transcriptomic and epigenomic data quality. Keep the nuclei aliquot intended for downstream tagmentation on ice at all times.
  7. When recovering nuclei from the Percoll‱ gradient, aspirate slowly and avoid disturbing the lower layers. Accidentally collecting the lower dark green plastid-rich band will increase plastid contamination.
Before start
  1. If this protocol is being used only to optimise nuclei isolation and the isolated nuclei will not be used for downstream single-nucleus applications, such as 10x Genomics Single Cell Multiome, replace the RNase inhibitor with nuclease-free water. Protector RNase Inhibitor is expensive and should be reserved for preparations intended for downstream RNA-based single-nucleus workflows.
  2. On the day of a single-nucleus Multiome experiment, do not deviate from the recommended brand or working concentration of Protector RNase Inhibitor. Using the wrong RNase inhibitor, or omitting it, can severely compromise RNA recovery.
  3. Prepare all buffers fresh on the day of the experiment. Add Protector RNase Inhibitor (Sigma Cat. No. 3335399001) immediately before use. Prepare the 30% and 70% Percoll‱ solutions using Wash Buffer without RNase inhibitor, as the inhibitor concentration is already accounted for in the Percoll‱ recipes.

Mechanically releasing nuclei into solution
18m 50s
The following volumes are for a single nuclei isolation. Scale up accordingly.
Weigh 180 mg fresh green tissue and place into a small plastic Petri dish on ice.
10m
Add 400 µL Wash Buffer containing 1 U/µL Protector RNase Inhibitor.
10s
Chop the tissue on ice for a maximum of 2 min using a new razor blade, such as a Feather platinum-coated double-edge blade.
Note: Do not over-chop the tissue. The aim is to produce a slurry-like consistency. Excessive or overly rapid chopping can damage nuclei integrity.
2m
Add an additional 350 µL Wash Buffer and incubate the lysate on ice for 2 min. Gently agitate the Petri dish to evenly distribute the chopped material in the buffer.
2m
Tilt the Petri dish to approximately 45° and collect the liquid lysate using a wide-bore P1000 pipette tip.
30s
Filter the lysate through a pre-wetted 20 µm mini PluriStrainer into a 2 mL Eppendorf tube.
30s
Centrifuge the filtrate at 50 × g for 3 min at 4°C using a swinging-bucket rotor to pellet large debris, including non-glandular trichomes.
3m
Carefully aspirate 500 µL supernatant using a P1000 pipette, taking care not to disturb the loosely packed debris pellet.
10s
Filter the supernatant through a new pre-wetted 20 µm mini PluriStrainer into a fresh 2 mL Eppendorf tube.
30s
Densit gradient centrifugation
16m 30s
Using a P200 pipette, carefully underlay 200 µL 30% Percoll‱ beneath the filtered nuclei suspension.
30s
Carefully underlay 200 µL 70% Percoll‱ beneath the 30% Percoll‱ layer.
30s
Centrifuge at 2,500 × g for 15 min at 4°C.
15m
Carefully aspirate 200 µL from the green band formed at the interface between the Wash Buffer and 30% Percoll‱ layer.
Note: This fraction contains high-quality intact nuclei, with minor chloroplast contamination. Use a P200 fitted with a wide-bore pipette tip for better flow control and avoid disturbing the lower green band at the 30% 70% interphase layer.
30s
Plastid lysis and nuclei permeabilization
28m 30s
Add 1 mL Lysis Buffer to the collected nuclei fraction.
10s
Mix by gently inverting the tube and incubate on ice for 5 min, gently inverting once every minute.
5m
Pass the suspension through a pre-wetted 20 µm cell strainer using Lysis Buffer.
30s
Centrifuge at 500 × g for 5 min at 4°C.
5m
Carefully remove the supernatant without disturbing the nuclei pellet.
10s
Resuspend the pellet in 1 mL Wash Buffer if the pellet is translucent, if the pellet is green then resuspend in 1mL Wash Buffer containing 0.2% Triton X-100. Mix gently by pipetting 5 times.
30s
Centrifuge at 500 × g for 5 min at 4°C.
5m
Carefully remove the supernatant.
10s
Repeat wash twice more using 1 mL Wash Buffer without Triton X-100.
6m
Centrifuge at 500 × g for 5 min at 4°C.
5m
Carefully remove the supernatant without disturbing the nuclei pellet.
30s
Resuspend the nuclei pellet in 50 µL Diluted Nuclei Buffer by gently pipetting 5 times.
30s
Nuclei counting and quality control
11m 30s
Immediately transfer 20 µL nuclei suspension to a new RNase-free, low-bind tube for downstream tagmentation. These nuclei should not be stained with DNA binding dyes. Keep this tube on ice.
10s
Transfer 20 µL of the remaining nuclei suspension to a separate tube for microscopy.
10s
Add 1 µL Hoechst 33342, 50 µg/mL, and gently mix by pipetting 5 times with a P20 pipette.
10s
Load 10 µL stained nuclei suspension onto a haemocytometer.
1m
Count nuclei and assess nuclei integrity by microscopy.
10m
If nuclei yield, integrity and cleanliness are acceptable, proceed immediately to the 10x Genomics protocol for nuclei dilution, if required, and tagmentation.
Load 10 µL of stained nuclei suspension onto a haemocytometer. Count nuclei and assess nuclei integrity, debris content and clumping by microscopy. If large nuclei clumps (>5 nuclei per aggregate) dominate the preparation, adjust the experimental parameters as described in Troubleshooting. Alternatively, pass the remaining nuclei suspension through a 10 µm or 20 µm cell strainer and reassess the filtrate to confirm removal of large clumps and retention of sufficient high-quality nuclei for downstream single-nucleus library generation.

Isolated nuclei at 40X magnification. Nuclear membrane should be intact in >70% nuclei and minimal clumping should be observed.

Following a successful outcome of step 20. Proceed to 10x protocol for nuclei dilution and transposition.
snRNA + snATAC sequencing library fragment quality control

Representative D5000 TapeStation traces of amplified cDNA from Cannabis sativa inflorescence nuclei prepared using the 10x Genomics Multiome workflow. Profiles are shown for control and methyl jasmonate-treated biological replicates. Libraries displayed the expected broad amplified cDNA size distribution, indicating successful reverse transcription and cDNA amplification prior to gene-expression library construction.

Representative D5000 TapeStation traces of final gene-expression sequencing libraries generated from Cannabis sativa inflorescence nuclei using the 10x Genomics Multiome workflow. Profiles are shown for control and methyl jasmonate-treated biological replicates. Libraries showed the expected compact fragment-size distribution for 10x gene-expression libraries and were suitable for sequencing.

Representative D5000 TapeStation trace of amplified Cannabis sativa ATAC-seq libraries. Libraries show the expected ATAC-seq fragment distribution, including a low-molecular-weight/nucleosome-free fraction and visible nucleosomal periodicity, indicating successful tagmentation and library preparation.

Technical optimisation of transposition time for smaller genomes when using 10X snATAC or snMultiome
The standard 10x Genomics transposition time guideline may require empirical optimization for smaller genomes as it was optimised using mouse and human genomes (~3 Gb) while the cannabis genome is significantly smaller (~800 Mbp). When working with new species and variable genome sizes titration of Tn5 transposition time is a standard pre-optimisation step in bulk ATAC work flows.

In a separate experiment using the same protocol and reagents we made snATAC-seq libraries derived from tomato, a species with a genome size very similar to Cannabis sativa. In this instance we reduced the transposition incubation time by 10 min. This showed a marked improvement in the fragment-size profile and produced clearer nucleosomal periodicity. We note that technical and biological variables may have confounded this observation and should therefore be interpreted cautiously. To this end we highlight the need for empirical studies of transposition optimisation on a wider range of genome sizes in commercial snATAC workflows. Despite this, our optimization supported the use of shortened transposition conditions for smaller genomes rather than the use transposition time more suited for mouse and human genomes.

Representative tomato 10X Genomics snATAC-seq TapeStation traces used during transposition-time optimization. Reduced transposition time improved the amplified library fragment distribution and nucleosomal periodicity relative to the original transposition time.

Protocol references

Citation
Sikorskaite S, Rajamäki ML, Baniulis D, Stanys V, Valkonen JP (2013). Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families. Plant methods.
https://doi.org/10.1186/1746-4811-9-31
LINK

Citations
Sikorskaite S, Rajamäki ML, Baniulis D, Stanys V, Valkonen JP. Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families.
https://doi.org/10.1186/1746-4811-9-31