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Sample Collection and Storage by Sample

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General Protein Storage(“Protein Purification,” 2022) For short-term storage (~24h), most proteins can be kept at 4°C. For long-term storage, protein samples are typically kept at -20°C or -80°C. Protein storage at -20°C usually requires the addition of 50% glycerol to your sample to avoid freezing at this temperature. If we plan to store a protein at -20°C, we generally run the final size exclusion chromatography step in 2x storage buffer and then dilute the sample 1:1 with 100% glycerol. Alternatively, the protein sample can also be dialysed against the storage buffer already containing 50% glycerol. Proteins stored at -20ºC are often stable for several months, although the exact time frame is protein-dependent and should be determined experimentally. Protein samples stored at -80ºC will be frozen. As repeated freeze-thaw cycles usually have a negative influence on protein samples, it is best to prepare small-sized, single-use aliquots that will be used up during the course of an experiment. 5-10% glycerol or other additives that protect against the effect of freezing and thawing can be added as well. After preparing your protein sample aliquots, it is important to flash-freeze them in liquid nitrogen before importing them into the -80ºC freezer for long-term storage. Many proteins are stable for months to years when stored under appropriate conditions at -80°C, but the exact time frame varies from protein to protein and should be determined experimentally.

Sponges(Ruocco et al., 2021) – washed three times with filter-sterilised natural seawater, one fragment placed in 70% ethanol, one fragment placed in RNALater, stored at -20°C

Seawater(Acinas et al., 2021) – filtered through a 200 µm, then a 20 µm mesh, then vacuum filtered through a 0.8 µm filter, then a 0.2 µm filter, flash frozen in liquid nitrogen & stored at -80°C (all done with ~4h; water at 4°C during processing)

Seawater(Michoud et al., 2021) – filtered through a 0.2 µm filter, filter stored in 2 ml tubes containing lysis buffer (EDTA 40 mmol l−1 pH 8.0, Tris-HCl 50 mmol l−1 pH 8.0, sucrose 0.75 mol l−1) flash frozen in liquid nitrogen, filtered & unfiltered water stored in 15 ml falcons at -20°C

Prokaryotes in sea water(Baltar et al., 2018) – water sampled without filtration, 2% final concentration glutaraldehyde added, stored at 4°C for 15 min in the dark, flash-frozen in liquid nitrogen and stored at -80°C

Swedish cervical biobank(Perskvist et al., 2013) – stored in liquid at -25°C, using liquid-based cytology cells suspended and fixed in Thinprep (TP) containing 20 ml PreservCyt (ThinPrep Hologic, Boxborough, MA) 4 ml of liquid-based cytology sample from the bottom of the patient tube is transferred into a conical tube and allowed to sediment for 30 min 300 µl of the sediment are transferred to a cryotube and stored at -25°C

Pooled tear collection(Willis et al., 2020) – centrifuged at 270 g for 3 min, supernatant stored in 1.5 ml microcentrifuge tubes at -80°C

Mosquitos(Ali et al., 2021) – human bait adult catching method, pool 3-5 mosquitos, store at -80°C

Oral swabs(Gao et al., 2020) – after collection swab is immersed in 10:1 Tris-EDTA buffer immediately and stored at −80 °C

Fruit fly larvae(Majumder et al., 2020) – sterilise larvae with a solution of 0.5 % Tween 80, 0.5 % sodium hypochlorite and 80 % ethanol for 30 sec, rinse them 3 times in PBS for 30 sec, use sterile pestle to crush larvae and store in BHI containing 20 % glycerol at -80°C

Turkey Trachea swabs(Kursa et al., 2021) – placed in 1 ml PBS, then stored at -20°C

Frog toes(Boyle et al., 2004) – stored in sterile 1.5 ml tubes at -80°C

Leaves(Humphrey & Whiteman, 2020) – surface sterilise: rinse for 5 sec 95% ethanol, 30 sec in 70% ethanol, 30 sec in 10% bleach, followed by three 2-minute washes in sterile water, air-dry for 5 min, then add to 2 ml tube containing 350 µL 10 mM MgSO4 and a sterile 5 mm steel ball and homogenise using a TissueLyser (QIAGEN) run at max speed (50 Hz) for up to 60 sec, then flash freeze in liquid nitrogen and store at -80°C

Atlantic salmon(Karlsen et al., 2022) – dissection samples placed in RNAlater and stored at -80°C

Yoghurt(Islam et al., 2021) – after purchase, samples were placed immediately at 4°C for transport and within 12 h stared at -80°C

Activated sludge from wastewater(Morin et al., 2020) – collected samples stored at 4°C for transport, within 6 h centrifuged at 6 500 g for 15 min at 4°C, supernatant removed and pellets stored at -20°C

Penguin cloacal swaps(Wille et al., 2020) – placed in tubes containing viral transport medium (brain heart infusion [BHI] broth-based medium [Oxoid] with 0.3 mg/ml penicillin, 5 mg/ml streptomycin, 0.1 mg/ml gentamicin, and 2.5 μg/ml amphotericin B), kept on ice for 4 hours, then stored at -80°C

Antarctic ticks(Wille et al., 2020) – collected from rocks, placed in RNALater and stored at -80°C within 4-8 h of collection

Frog swabbing(Ellison et al., 2021) – rinse caught frog with 50 ml sterilised water to remove transient bacteria, swab entire skin surface with a sterile Dacron swab for 30 sec, place swab in microcentrifuge tube containing PBS and store on ice, then transfer to -80°C storage

Invertebrates (in-house protocol provided by Justine Vandedorpe) – whole samples are stored in 80 % ethanol, in fridge for short-term storage, and in -20°C freezer for long-term storage.

Hooded seal brain (in-house protocol provided by Justine Vandedorpe) – whole samples are placed in 4°C glucose artificial cerebrospinal fluid (aCSF) saturated with 95 % O2 - 5 % CO2. After experiment (induced normoxia and hypoxia), tissues were stored in RNAlater stabilisation fluid (Life Technologies, USA) and kept at 4°C.

Saliva(in-house protocol provided by Pamela Ferretti) - passive drooling procedure in sterile 50 ml screw top tubes (approximately 3 ml of saliva per sample). About 30 minutes before collection, all volunteers performed a mouth rinse with drinking water. Samples were vortexed before 2 ml of each was centrifuged at 10,000 × g for 10 minutes to collected the bacterial pellets. Total DNA was extracted using the PowerSoil DNA Isolation Kit (MoBio), according to manufacturer’s instructions. DNA was stored at −20 °C until used.

Skin swab(in-house protocol provided by Pamela Ferretti) - Skin samples were collected using Catch-All-Swabs (Epicentre Technologies, Wisconsin, USA) shortly after birth but before the skin-to-skin contact with the infant, by swabbing the upper area of the maternal breast (intermammary cleft). After pre-moistening with 2 ml SCF-1 buffer (50 mM Tris buffer, pH 7.6, 1mM EDTA, pH 8.0, and 0.5% Tween-20) (Human Microbiome Project Consortium, 2012) contained in a 15 ml sterile screw top collection tube (Sarstedt, Nümbrecht, Germany), the swab head was rubbed back and forth for approximately 30 seconds over the area (repeating twice) before the swab was returned to the buffered solution. The lower part of the swab was broken to ensure closure of the tube (see below). After sampling, deterge the sampled area with a clean pad of cotton and water.

Vaginal swabs(in-house protocol provided by Pamela Ferretti) - Skin samples were collected using Catch-All-Swabs (Epicentre Technologies, Wisconsin, USA). The swab was rubbed 5 times, with a circular motion, in the vaginal introitus and then the swab head was placed in a 15 ml sterile screw top collection tube containing 2 ml SCF-1 buffer (see photos above).

Tongue dorsum swabs(in-house protocol provided by Pamela Ferretti) – Skin samples were collected using Catch-All-Swabs (Epicentre Technologies, Wisconsin, USA). Samples were collected by rubbing a swab on the central area of the back of the tongue for approximately 5 seconds. The swab head was then placed in a 15 ml collection tube containing 2 ml SCF-1 buffer (see photos above). Samples were collected while wearing protective disposable gloves to avoid skin contamination.

Maternal Milk (without glycerol)(in-house protocol provided by Pamela Ferretti) Minimum of 2ml of milk was self-collected by the mothers in sterile falcon tubes while wearing disposable gloves. No buffer was added. Samples were stored at -4C right after collection and moved to -80C within a week. Note that for colostrum and other milk samples within the first week of life the 2ml min quantity might not be possible. In that case, the mother collected whatever quantity was possible in 10 min timespan.

Maternal Milk (with glycerol)(in-house protocol provided by Pamela Ferretti) Minimum of 2ml of milk was self-collected by the mothers in sterile falcon tubes with added 20% glycerol while wearing disposable gloves. Samples without were stored at -20C for cultivation experiments. Note that for colostrum and other milk samples within the first week of life the 2ml min quantity might not be possible. In that case, the mother collected whatever quantity was possible in 10 min timespan.

Stool samples(in-house protocol provided by Pamela Ferretti) – samples were self-collected using collection tubes specific for faecal material (Sarstedt, Nümbrecht, Germany). Toilet paper was placed at the bottom of the WC, to prevent stool samples from sinking and getting contaminated. The collection was performed at the upper part of the feces, the one not in contact with the toilet paper, WC walls or other material. Subjects were instructed, whenever possible, to urinate before evacuating the stools. Samples were collected in variable quantity, depending on availability (meconium is usually only one spoon). Once collected the tube was placed at -4C as soon as possible, and then transferred to -80C within a week.

Faeces/gut content (in-house protocol provided by Johannes Masson) – flash freeze in liquid nitrogen and store at -80°C (dissolve in Stool DNA Stabilizer (INVITEK Molecular) while thawing; or if you have more time dissolve in Stool DNA Stabilizer right after collection and normally freeze and store at -80°C)


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