Methods derived from Duruz et al. 2023. [@DuruzLoligoSingleCell2023]

Reagents

Papain enzyme solution

• Papain (Sigma #P3125)
• Concentration 1 mg/ml in nuclease-free PBS

Protocol

1. Embryos were first dissected out of their eggs in stages 28–30 (pre-hatchling stage) and their heads were isolated and put in nuclease-free phosphate buffer (PBS) on ice. The heads were then
2. Centrifuge for 5 min at 4°C
3. Carefully remove supernatant and replace with papain enzyme solution solution
4. Incubate tissue for 30 min at room temperature (20–25°C) under continuous agitation.
   • During the incubation period, gently triturate up and down every 10 min to facilitate dissociation.
5. Centrifuge cell suspension at 2000 rpm for 5 min at 4°C.
6. Remove supernatant replace with 1 ml of PBS containing 0.04% bovine serum albumin (BSA) to stop the enzymatic reaction.
7. Repeate previous wash step one more time to ensure removal of the enzyme.
8. Filter suspension through a 40 μm Flowmi Cell strainer (Bel-Art H13680-0040).
9. Spin at 2000 rpm for 5 min at 4°C.
10. Discard supernatant, and resuspend cells in 100 μl of PBS + 0.04% BSA with added RNAse inhibitor (0.1 μL/ml) and gently pipette the entire volume approximately 200 times to ensure dissociation.
11. Assess cell concentration and viability via countess or similar method with PI or Acridine Orange (AO) assay.

References

[^ref]

Questions

• ASW instead of PBS?
• TryplE instead of papain?
• Trehalose to stabilize membranes?
• Addition of RNAse inhibitor everywhere?