Larvae dissociation

Paganos P, Voronov D, Musser JM, Arendt D, Arnone MI. Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome. Elife. 2021 Nov 25;10:e70416. doi: 10.7554/eLife.70416.

Dissociation protocol

1. Collect and concentrate the larvae using a 40 μm mesh filter.

• The concentration of the larvae is one of the most crucial steps. Make sure you end up having a pellet of 100-200 μl of larvae.
• Before starting with the dissociation process pre-chill Calcium and Magnesium free Artificial Sea Water (Ca2+ Mg2+free ASW) as well as the dissociation buffer.

2. Transfer the larvae into a 1.5 ml low binding eppendorf tube.

3. Spin down the larvae at 500g-700g for 5-10 minutes at 4°C (optimally in a swing centrifuge) and remove seawater.

4. Resuspend larvae in 600 μl of Ca2+ Mg2+free ASW.

5. Spin down the larvae at 500-700g for 5-10 minutes at 4°C and remove Ca2+ Mg2+free ASW.
Note: (Optional) Repeat the steps 4 & 5 one more time.

6. Resuspend larvae in 500 μl of dissociation buffer and incubate on ice for 10 min (every 2 min pipette up and down).

Notes:

• For the pipette aspiration step use a P1000 set to approx. 900μl. In this way all the liquid from the tube will be going in and out of the tip every time you pipette up and down.
• It is important to find a balance betweentoo violent and too gentlepipetting. You have topipette strong enough to apply mechanical force to promote dissociation but not too strong that will result in lysis of the cells.

Monitor the progress of the dissociation by periodically examining a drop of the suspension (about 10 μl). Whenever you are not pipetting leave the samples on ice.

1. Early embryonic stageswill dissociate fasterthan the larval ones. It is possiblethat by the end of the 10 min incubation larvae will be fully dissociated so step 7 can be skipped.

7. Once dissociation is complete, spin down the single cells at 700g for 10 minutes at 4°C to remove the dissociation buffer.

Notes:

1. From this stage and onwards it will be harder to pellet the single cells. Depending on the amountof larvae you initially used you can decidewhether it’s worth it losing a few cells or centrifuging for longer time.
2. Resuspension of the cells in normal artificial sea water should be avoided (containing calcium/magnesium) since it will immediately promote cell aggregation.

8. esuspend the single cells in 100 μl of Ca2+ Mg2+ free ASW.

9. To remove aggregated cells, pass the specimen through a 40μm cell strainer. To do so place the strainer on top of a clean 1.5 ml low binding eppendorf tube. Place the sample in the middle of the strainer and let it pass through. In case the drop containing cells cannot pass through, use a P1000 pipette to manually collect it from the other side of the strainer.

Solution recipes

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