Seagrass Collection & Shipping Protocol

Purpose

This document tells a collector three things: what species to prioritize, where the useful populations are, and how to handle and ship the tissue so it arrives frozen and sequence-ready. It pairs with the gDNA sampling steps (Section 5), which are unchanged from the field draft.

The sequencing goal is two data types per sample: a whole genome and a methylome (DNA methylation map). Both come from the same frozen leaf tissue, so a single clean collection serves both. Consistency of sampling matters more than volume — see the handling rules before going out.

Collection priorities

We are collecting where the public record is thin and the biology is most useful. Three tiers, in order:

Candidate sites at a glance

Regions are drawn from OBIS/GBIF occurrence records and mark where the species is common, not specific permitted sites. Confirm access, collection permits, and any CITES/phytosanitary requirements for the specific location before sampling — this is especially important for international (Mexico, Mediterranean) collections.

How much to collect

• Per site: aim for 8–12 individual plants, sampled far enough apart (meters) to avoid resampling the same clone. Seagrass spreads clonally, so nearby shoots may be genetically identical; spacing captures real genetic diversity.
• Per plant: one 2–3 inch leaf-tip segment is enough for both genome and methylome. A second segment in a separate tube is useful insurance if space and dry ice allow.
• Label every tube with site, plant number, date, and collector before freezing.

Specimen handling (gDNA sampling)

These steps are the field-tested protocol; follow them exactly. Consistency between plants is the single most important factor for clean methylome data.

1. Before sampling, collect materials: screw-cap tubes with labels on the side, forceps, scissors, a plastic freezer bag, a styrofoam box, and dry ice.
2. Cut 2–3 inches of leaf tissue with the scissors.

• Be extremely consistent across plants: sample roughly the same tissue age (don't mix an old damaged leaf from one plant with a young developing leaf from another) and the same part of the leaf (base vs. tip).
• Recommended: take the tip of an undamaged leaf.

1. Fold the tissue and use the forceps to stuff it into a screw-cap tube.
2. Place the tube in the plastic freezer bag — one bag holds all samples from a site; it makes the tubes easy to find in the lab.
3. Cover the tube in dry ice as quickly as possible for fast, even freezing. The faster tissue freezes, the better the DNA and methylation signal are preserved.
4. Keep frozen until shipment. Do not let samples thaw between collection and the lab.

Packaging & shipping

The rule is simple: the samples must stay frozen from the field to the lab bench. Dry ice sublimates (turns to gas, roughly 1 kg lost per 24 h from a typical cooler), so the whole process is built around keeping enough of it packed around the tubes and getting the box to the lab fast.

Safety first

Dry ice is −78 °C and turns directly into CO₂ gas. Two hazards to respect at every step:

• Cold burns. Always handle dry ice with insulated gloves and tongs — never bare skin. Wear eye protection when breaking up blocks.
• Suffocation risk. Subliming dry ice displaces oxygen. Work in a ventilated space, never in a sealed car cabin or small closed room, and never store the cooler in an airtight container.

Packing the box — step by step

1. Confirm samples are already frozen. Every tube should have been snap-frozen in dry ice at collection (handling step 5). Do not pack tubes that have warmed or thawed.
2. Line the styrofoam cooler. Use a hard-walled styrofoam box (or a styrofoam insert inside a cardboard carton). Put a base layer of dry-ice pellets (2–3 cm) across the bottom.
3. Add the bagged tubes. Place the freezer bag(s) of labeled tubes on the base layer. Keep the tubes together so they're easy to find on receipt.
4. Bury the tubes in dry ice. Surround and cover the tubes completely with dry-ice pellets — tubes in the middle of the ice, never sitting on top. Pellets pack more densely around small tubes than block ice; if only block ice is available, break it small. The goal is even, all-around cold.
5. Fill void space. Top up with dry ice and/or crumpled packing paper so the tubes cannot shift during transit. A partly empty box lets samples slide into a warm corner.
6. Do not seal airtight. Subliming CO₂ must be able to vent — leave the inner bag open or loosely closed, and use a styrofoam lid (not a vacuum seal). A sealed container will build pressure and can rupture.
7. Close and tape the outer carton, leaving the styrofoam vented. The outer cardboard can be taped normally; the venting happens through the styrofoam seams.

Amount and timing

• How much dry ice: carriers cap dry ice at 2.5 kg (5.5 lb) per package for shipments on passenger aircraft, which is what most overnight air uses. Stay at or under that limit — about 2 kg in a small styrofoam shipper is enough to keep a dozen tubes frozen through a 24-hour overnight transit. There's no need to overpack: a small, well-insulated box with ~2 kg beats a large box with more.
• Top off at the last moment. Add a fresh layer of dry ice right before carrier pickup, not the night before.
• Ship early in the week (Mon–Wed) and overnight whenever possible, so a package never sits in a facility over a weekend. A 2-day ground shipment will usually arrive thawed.

Labeling and regulations

Dry ice is a regulated shipping material — UN1845, IATA/DOT Class 9 (miscellaneous dangerous goods):

• The outer box needs a Class 9 dry-ice hazard label and the UN1845 marking, with the net weight of dry ice in kg written on it.
• The airway bill / shipping manifest must declare dry ice and its weight (see the per-package limit above).
• Most carriers (FedEx, UPS) handle dry-ice shipments routinely — tell them it contains dry ice when you book, and they'll apply the correct service and paperwork.
• Frozen plant tissue itself needs no special permit for domestic US shipment. International shipments (Mexico, Mediterranean) additionally require phytosanitary / import clearance — arrange this before collecting, since it can take time.

Ship to

Send frozen samples to Decibel Bio's lab, topped off with dry ice, overnight where possible:

Decibel Bio
c/o Brandon Pfannenstiel
2625 Durant Avenue
Berkeley, CA 94720

Before shipping, email Brandon (brandon@decibel.bio) the expected carrier, tracking number, and delivery date, plus a quick manifest (species, sites, number of tubes) so the lab can receive and re-freeze the samples immediately on arrival. Avoid shipments that would arrive on a weekend unless arranged in advance.

Quick field checklist

• [ ] Permits / site access confirmed (and phytosanitary clearance for international sites)
• [ ] Materials: labeled screw-cap tubes, forceps, scissors, freezer bags, styrofoam shipper, dry ice (~2 kg; ≤2.5 kg per package), insulated gloves
• [ ] 8–12 spaced plants per site; one labeled leaf-tip tube per plant
• [ ] Consistent tissue age and leaf position across all plants
• [ ] Tubes frozen in dry ice immediately, kept frozen
• [ ] Box packed with tubes buried in dry ice, vented, Class 9 / UN1845 labeled
• [ ] Overnight shipment booked; tracking + manifest emailed to Decibel before pickup