Jun 26, 2026
Version 5
Apple Leaf Field Collection Protocol for Enzymatic Methyl-seq V.5
- Thomas Maret1,
- Anthony Venon2,
- Amandine Cornille2
- 1Génétique Quantitative et Évolution – Le Moulon, Université Paris-Saclay, INRAE, CNRS, AgroParisTech, 91190 Gif-sur-Yvette – France;
- 2Division of Science, New York University Abu Dhabi – Saadiyat Island, Abu Dhabi – United Arab Emirates
Protocol Citation: Thomas Maret, Anthony Venon, Amandine Cornille 2026. Apple Leaf Field Collection Protocol for Enzymatic Methyl-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2dx8jg1y/v5Version created by Thomas Maret
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: June 26, 2026
Last Modified: June 26, 2026
Protocol Integer ID: 319842
Keywords: Field Sampling, Apple Leaf Collection, EM-seq, seq dna methylation analysis, apple leaf collection for em, collecting apple leaf, apple leaf collection, apple leaves for em, seq this protocol, sampling, seq, pooled healthy leaf, sample quality, healthy leaf, peak photosynthesis, field sampling protocol, sample, dna, apple leaf field collection protocol for em, apple leaf field collection protocol, enzymatic methyl
Funders Acknowledgements:
Tamkeen, NYU Abu Dhabi Research Institute
Grant ID: Grant AD 454
19 Washington Square North New York University
Abstract
This protocol describes a standardized method for collecting apple leaves for EM-seq DNA methylation analyses. Sampling is performed within a controlled time window around peak photosynthesis, using pooled healthy leaves preserved immediately at low temperature to ensure sample quality and consistency across sites.
Materials
- Leaf punch (cork borer or biopsy punch), 3–6 mm in diameter
- 2 mL Eppendorf tubes (one tube per tree), pre-labelled
- 70% ethanol (for cleaning the punch between trees)
- Coloured ribbons (to pre-mark the trees that will be sampled twice)
- Orchard map showing the trees to be sampled, including those sampled twice
- Sample preservation system, one of the following (in order of preference):
- Liquid nitrogen
- Dry shipper charged with liquid nitrogen
- Cooler box with ice (only if neither LN_2 nor a dry shipper is available)
- Permanent marker / labels for tube identification
- Field notebook for recording tree ID, site, date, and sampling time
Background and objective
This protocol describes the field sampling procedure for collecting apple leaves to be used in an EM sequencing (EM-seq) experiment. The same protocol is used for wild and domesticated trees.
Materials and equipment
- Biopsy punch, 3–6 mm in diameter
- Cutting mat or cutting board
- Microcentrifuge tubes 2 mL
- Stainless steel beads, 4 mm in diameter
- Paper envelopes
- Plastic zip-lock bags
- Silica gel beads
- Ethanol 70%
- Distilled water
- Absorbent paper
- Small plastic pots
- Sample preservation system, one of the following, in order of preference:
- Liquid nitrogen
- Dry shipper charged with liquid nitrogen
- Cooler box with dry ice
- Permanent marker
- Field notebook
Sampling window, order and time tracking
Leaves must be collected around the daily maximum of photosynthesis. Sampling window: 1 h 30 min before to 1 h 30 min after the sun culmination (total window of 3 hours). All samples for a given tree must be collected within this window so that the physiological state of the leaves is standardized across sites. Use a solar time calculator (e.g. SunCalc).
Sample the orchard line by line, following the rows of the orchard. This ordered sampling is critical because DNA methylation varies with the circadian cycle, and a consistent line-by-line order allows to account for these methylation changes during downstream analysis.
Write the current time on every envelope once it has been filled with punched leaves. You can also note any relevant observations (weather, leaf condition, pests, etc. ) in a field notebook.
Before the sampling date
- Wash the stainless steel beads with soap and water, rinse very well with distilled water. Dry the beads in an incubator at 70°C for at least one night. Autoclave the clean beads at 121°C for 20 min. Another drying step overnight at 70°C can be needed after the autoclave.
- Fill the 2 mL microcentrifuge tubes with two stainless steel beads. Label the tubes according to the trees sampled. Each tree is sampled once, so you only need one tube per tree.
- Label the envelopes in the same way, one labelled tube correspond to one labelled envelope.
- Sort the envelopes in a plastic zip-lock bag with silica gel beads.
Sampling procedure (per tree)
- Confirm the tree identity. Sample strictly line by line.
- Collect 9 leaves distributed around the tree, all taken from the mid-canopy. Choose only healthy leaves free of visible pathogens (no spots, mildew, rust, necrosis, or heavy insect damage).
- Stack the 9 leaves on top of each other and punch all of them at once with the biopsy punch. Eject the 9 leaf discs directly into the pre-labelled 2 mL microcentrifuge tube assigned to that tree.
- Close the tube tightly and immediately transfer it to liquid nitrogen, a dry shipper (LN₂-charged), or dry ice, depending on the preservation system available at the site, in this order of preference.
- Place the 9 perforated leaves in the paper envelope that corresponds to the collected tree.
- Write the current time on the envelopes.
- Note any relevant observations (weather, leaf condition, pests, etc.).
- Before moving to the next tree, clean the punch by soaking it in a pot with 70% ethanol to prevent cross-contamination between trees. To rinse it, use a second soaking pot with distilled water. Dry it with absorbent paper.
- Move to the next tree on the same line. Only start a new line once the current line is complete.
Quality control notes
- Use a freshly cleaned punch for each tree.
- Never share a tube between trees.
- Avoid leaves showing pathogen symptoms (spots, mildew, rust, necrosis) and leaves with heavy insect damage.
- Keep tubes cold (LN₂, dry shipper, or dry ice) from the moment of collection until long-term storage.
- Sample strictly line by line and record time on every envelope. This is required to correct the analysis for circadian methylation effects.
- The dried leaves in the envelopes can be used for biochemical or phenotypic analyses.
- Be aware of the risks of exposure to liquid nitrogen and ethanol, and ensure that appropriate personal protective equipment (PPE) is worn at all times.
- If wanted, photograph each sampled tree and store the photo with the same ID as the tube.