Sample Guide

Collection notes

• Keep sample type and anticoagulant consistent within a project; never mix serum and plasma
• Diet affects metabolites — standardise fasting time for clinical samples and record it
• Avoid buffers with detergents or high salt, which interfere with chromatography
• Aliquot into single-use volumes to eliminate repeat freeze–thaw

Note: targeted assays size the sample by the analyte list; low-abundance compounds may need larger volumes — confirm at design stage.

Step-by-step: serum / plasma

1. Draw after 8–12 h fasting; keep the anticoagulant consistent (EDTA or heparin) — use a clot activator tube for serum
2. Serum: stand at room temperature for 30 min. Plasma: mix gently 5–8 times immediately
3. Centrifuge at 3000 g for 10 min at 4 °C and transfer the supernatant, avoiding the buffy coat and any haemolysis
4. Aliquot 100–200 μL per tube, label with ID and time, move to −80 °C within 2 h
5. Ship on dry ice, keeping below −70 °C; dry ice must not run out before arrival

Step-by-step: tissue and cells

1. Rinse tissue in pre-chilled PBS within 5 min of collection; remove fat and connective tissue
2. Cut into pieces of ≤ 50 mg, snap-freeze in liquid nitrogen for at least 30 s, then store at −80 °C
3. Wash cells twice with pre-chilled PBS to remove medium (residual serum interferes with chromatography)
4. Aspirate the supernatant and snap-freeze the pellet — do not freeze cells in PBS

Common mistakes

• × Mixing serum and plasma or changing anticoagulants → groups are not comparable
• × Repeated freeze–thaw of one aliquot → metabolite degradation and poor reproducibility
• × High-salt or detergent-containing buffers → ion suppression and column contamination
• × Using haemolysed samples for targeted assays → falsely elevated intracellular analytes

Collection notes

• Design time points in groups (e.g. 0 / 15 min / 1 h / 4 h) and fix the sampling order in the protocol
• Quenching speed determines whether real flux is captured — process at low temperature immediately
• Always keep an unlabelled control from the same batch for natural-abundance correction
• Record tracer lot, concentration and addition time for reproducibility

Note: tracer, labelling duration and quenching are settled in a pilot run and define which pathways can be resolved.

Labelling workflow

1. Settle tracer concentration, labelling duration and quenching in a pilot run
2. At the target confluence, switch to fresh medium containing the tracer and record T0
3. Sample at each designed time point and quench each independently — never pool first
4. Collect the cell pellet and the medium separately, label and freeze
5. Always keep an unlabelled control group for natural-abundance correction

Key parameters

• Time points: at least 3–4 (e.g. 0 / 15 min / 1 h / 4 h) covering the rising phase, not only steady state
• Replicates: ≥ 3 biological replicates per time point, each started from an independent culture
• Quenching: liquid nitrogen or cold methanol (≥ 4 volumes) at −20 °C, all work on ice
• Records: tracer lot, volume added, culture temperature and exact sampling time

Common mistakes

• × Sampling all time points from one culture → the cells have already changed state
• × Labelling for 24 h → the system nears steady state and flux information is lost
• × Incomplete quenching → enzymatic reactions continue and the MID shifts

Collection notes

• Use low-binding tubes and wear gloves/mask to avoid keratin contamination
• Denature soon after lysis to limit protease degradation
• For PTM work keep matched controls and avoid phosphate buffers
• For PRM validation, provide the target protein list to guide method development

Note: protein samples are highly sensitive to freeze–thaw — aliquot so each tube is thawed once.

Sample processing

1. Cells: wash twice with PBS, then lyse directly (with protease inhibitors) or snap-freeze the pellet
2. Tissue: remove blood and connective tissue, weigh, add lysis buffer at 1:5–1:10 (w/v), homogenise on ice
3. Serum/plasma: avoid haemolysis; depletion of abundant proteins is decided per project
4. Centrifuge lysate at 12000–15000 g for 10–15 min at 4 °C and take the supernatant for quantification
5. Aliquot 100–200 μg per tube, store at −80 °C, ship on dry ice

Notes for PTM projects

• Phosphorylation: avoid phosphate buffers throughout; include matched controls
• Acetylation/ubiquitination: add deacetylase/deubiquitinase inhibitors and denature soon after lysis
• FFPE: state block age, fixation time and staining history; 4+ sections at 5 μm
• PRM: provide target protein/peptide lists to guide method development and RT confirmation

Common mistakes

• × No gloves/mask → keratin contamination masks low-abundance proteins
• × Lysates left at room temperature → protein degradation and loss of modifications
• × Estimating input from wet weight with high water/fat content → insufficient protein

Collection notes

• RNA integrity matters more than concentration — degraded RNA should not be sequenced
• Record sampling site, time and environment for microbial samples
• Use one library protocol within a batch to reduce batch effects
• Ensure dry ice lasts until delivery

Note: amplicon and metagenomic work is sensitive to contamination — use sterile consumables and include blank controls.

RNA workflow

1. Cut tissue into ≤ 5 mm pieces immediately, snap-freeze or immerse in RNAlater (4 °C overnight, then −20 °C)
2. Measure concentration and RIN; RIN ≥ 7 is required for library prep
3. Store RNA at −80 °C, avoid freeze–thaw, ship on dry ice
4. Keep a backup aliquot against shipping loss

Microbial and DNA workflow

1. Faecal/soil: collect ≥ 200 mg in a sterile tube, label, −80 °C within 2 h
2. Include a blank control handled with the same consumables to assess contamination
3. Whole blood: EDTA only — heparin inhibits downstream enzymatic steps
4. DNA: A260/280 within 1.8–2.0, avoid freeze–thaw and prolonged light exposure

Common mistakes

• × Submitting degraded RNA → library failure or 3′ bias
• × Faecal samples left at room temperature → community structure drifts
• × Different library batches within one comparison group → batch effects mask biology

Collection notes

• Suspension quality drives data quality: no clumps, minimal debris
• Keep dissociation short to avoid stress-response gene expression
• Do not store cells long in Ca²⁺/Mg²⁺-containing buffers
• If cryopreservation is required, confirm the protocol and check post-thaw viability

Note: run a small pilot to check viability and cell-type composition before committing to the full run.

Suspension preparation

1. Dissociate at low temperature, briefly and gently; target viability ≥ 80% with no clumps
2. Filter through a 40 μm mesh, centrifuge and resuspend in BSA-containing buffer
3. Adjust to 700–1200 cells/μL and record count and viability
4. Prepare fresh, transport at 2–8 °C and load within 4 h; avoid vigorous agitation

Key parameters

• Target cell number: load 1.5–2× the target capture number
• Keep dissociation ≤ 30 min; longer induces stress genes (Fos/Jun)
• Do not store cells long in Ca²⁺/Mg²⁺ buffers; never fix samples for live-cell marker staining

Common mistakes

• × Debris- and dead-cell-rich suspension → high empty-droplet rate
• × Loading hours after preparation → viability and transcripts degrade
• × Incomplete dissociation of large tissue pieces → distorted cell-type proportions

Collection notes

• Under- or over-fixation both mask antigens — follow the recommended fixation time
• For IHC with antibody sourcing, confirm species and clone in advance
• Do not fix samples before staining live-cell markers by flow
• State the required section orientation (transverse/longitudinal) on the submission form

Note: for pathology samples the sampling site drives interpretation — provide an H&E reference slide or imaging localisation where possible.

Per-assay workflow

1. ELISA: serum/plasma ≥ 50 μL, no haemolysis, aliquot into single-use volumes
2. WB: lyse on ice with protease inhibitors, quantify by BCA, aliquot ≥ 20 μg and denature
3. Flow: EDTA whole blood or suspension ≥ 100 μL, analyse within 24 h, list markers and isotype controls
4. Sections: fix in 4% PFA for 24–48 h (≤ 1.5 cm thick), dehydrate and embed in paraffin
5. Frozen sections: embed in OCT with the target face down, ship on dry ice, avoid freeze–thaw

Key parameters

• Fixation: too short and antigens are masked; over 72 h and epitopes are blocked
• Section thickness: 4–5 μm (paraffin), 8–10 μm (frozen) — confirm the purpose
• IHC with antibody sourcing: provide species, clone and expected localisation
• State section orientation and sampling site on the form; attach an H&E reference if possible

Common mistakes

• × Oversized tissue blocks → fixative cannot penetrate and the centre autolyses
• × Fixing before staining live-cell markers by flow → false negatives
• × Freezing samples without OCT → sections crack and crumble