Jill and I optimized lipid and metabolite tissue preparations for the E5 project. Detailed notes are in Jill’s notebook. Jill is now starting preparations of our samples for lipid and metabolite analyses at UW.

Description

Goals

Our goal was to adjust the protocol to make the following adjustments:

• Increase tissue slurry concentration by using less water during air brushing
• Remove interference from salts by airbrushing and preparing tissue homogenate in LC grade water
• Quantify protein content to estimate tissue input
• Prepare tube and box labeling plans
• Incorporate PBQC samples for lipid and metabolites

Activities

We did the following to accomplish the goals listed above:

Airbrushing

• We ran through the protocol with test samples.
• We changed airbrushing to airbrush with only air as much as possible and use water only when needed and to finish cleaning the skeleton.
• Airburshing is now done with LC grade water.
• We are using true eppendorf safe lock tubes and plastic pestles for homogenization.
• We tested host/symbiont fraction separations and confirmed symbiont pellet was removed with two rounds of centrifugation.
• We changed the airbrush from the vaccuum pump on the bench to an air compressor. This helped increase the pressure so that the concentration can be increased by using air without water.

Protein content

• We tested protein content of the test samples after separations and with revised airbrushing protocol.
• Protein concentrations were 700-900ug/mL.

Other preparations

• Jill located and isolated all the samples in preparation for processing.
• Ariana labeled all tubes and boxes.

Outcome

• At the end of the week we have a working protocol, all samples are located in the freezer, and tubes and boxes are prepared. We have obtained all required supplies.
• Jill is now ready to start tissue preparations from our samples.
• I emailed the UW facility with the protein values from our test samples, and the facility said they were in the optimal range of protein values. This indicates that we are providing enough tissue input for the analyses by increasing tissue slurry concentration.

Next steps

• When samples are prepared, we will send to UW. We will ask them to run a small set first to verify that signals are sufficient before proceeding with all samples.

• We will submit 96 samples for lipids, 96 for metabolites, and 2 PBQCs for each analysis.

Protocol

Working protocol for preparation of host tissue for lipidomic and metabolomic sample submission

1. Airbrushing and tissue quantification
2. Host symbiont separation
3. Sample preparation and shipping
4. Host protein

Equipment and Materials

• Ice / dry ice
• Weigh boat
• Scale
• Plastic pestles (can fit in 1.5 mL tube)
• 1.5 mL tubes
• 15 mL tubes
• 50 mL tubes
• Centrifuge
• Pipettes + tips
• Airbrush
• LC grade water

During ALL steps keep sample on dry ice and as cold as possible! Use ice cold LC grade water!

Airbrushing and tissue quantification

• When airbrushing, clean materials with 10% bleach, 70% ethanol and DI water in between samples. Keep all samples as cold as possible.
• Use ice-cold LC grade water.
• Thaw fragment on ice.
• Gently rinse fragment with DI water using transfer pipette to remove surface salts and seawater.
• Airbrush with AIR ONLY. Only use LC grade water to remove any remaining tissue at the end. This will ensure tissue is concentrated - we can add LC water later if needed to dilute.
• Pour slurry into 5-15 mL tube (smallest volume needed) and record total slurry volume
• Aliquot 1 mL of slurry into two 1.5 mL tubes per fragment. Label one of the tubes with a lipid number using a running serial number (i.e., L1-L96). Label one of the tubes with a metabolite number using a running serial number (i.e., M1-M96). Record metadata for each tube with Sample ID/Fragment ID. Label the side of each tube with the Sample ID and “L” for lipids or “M” for metabolites. Store in separate boxes.
• Aliquot 0.5-1 mL to a 1.5 mL falcon tube for protein measurements. Record volume of the aliquot taken. Return to the freezer. Return all samples to freezer if not proceeding with separation immediately.

Host fraction separation

• Homogenize each tube for one minute on ice using plastic pestle in tube to avoid lipid oxidation (do not use immersion homogenizer). Use a separate pestle for each fragment.
• Separate fractions by centrifuging at 2,500 g for 5 min at 4°C. Make sure to pre chill the centrifuge prior to adding samples.
• Move supernatant to new tube with the appropriate label and try to keep/scrape lipid films to keep with the supernatant. Avoid adding in any symbiont cells.
• Resuspend the symbiont pellet with 1 mL of LC grade water. Keep the tubes with symbiont pellets in a box labeled with “symbiont pellets”. Label the tube with “symbiont” if possible. Store in freezer for later use (in case we want to go back to them).
• Vortex the host fractions for 1 min and centrifuge again at 2,500g for 5 min at 4°C.
• Move supernatant to new tube, this is the host tissue; keep any lipid films as much as possible. Discard pellet if small or not visible - there will be few symbiont cells after this round.
• Add 10uL of each host sample into PBQC tubes. For “M” samples, add 10uL of host fraction to each of two PBQC-M tubes. For “L” samples, add 10uL of host fraction to each of two PBQC-L tubes.
• Return tubes to -80°C

Sample preparation and shipping

• Put one L tube in a box labeled “lipid samples” and one M tube for each fragment in a box labeled “metabolite samples”. We will send the UW facility the samples in these separately labeled boxes. The other tubes will be saved as back ups.
• Fill out sample metadata forms with tube number (M# or L#) and sample ID (informative sample ID for our use).
• Ship to UW on dry ice directly to the facility. Include sample metadata forms in the package. Use extra dry ice and insulated packages. Ship with fastest method.

Protein of host tissue

• Conduct BCA protein measurements for the HOST fraction for each sample. We will use these to check tissue input and perform any dilutions if needed.