Preparation of Protoplasts from Rice Inflorescences for Single Cell Sequencing

Preparation of Protoplasts from Rice Inflorescences for Single Cell Sequencing

1. Pre-treat the enzyme solution by heating it to 55°C for 10 minutes, cooling to room temperature, and then adding 10 mM CaCl₂ and 0.1% BSA (Sigma-Aldrich).
2. Tissue clipping: Take inflorescence tissue in good growth condition and quickly and gently cut it into small particles using a clean surgical blade.
3. Incubation: Incubate the tissue in 20% D-mannitol hypertonic solution for 5-10 minutes at room temperature in the dark to promote cell wall separation and facilitate subsequent cell wall degradation.
4. Blotting: Transfer the incubated tissue to clean filter paper and gently and quickly blot up the tissue outer epidermal solution.
5. Tissue digestion: Remove the cell walls at room temperature and prepare the protoplasts using an enzyme solution digestion system without RNase. Use the following reagent ratios:
Cellulose R-10
Macerozyme R-10
0.4 M mannitol
20 mM MES (pH 5.7)
20 mM KCl
10 mM CaCl₂
0.1% BSA
6. Perform microscopic examination of 5 μL at 1-hour intervals to monitor enzymatic digestion of the protoplasts.
7. Sieving: Use a 45 μm cell filter (Biologix, Biologix Group Ltd) to filter the protoplasts and take the suspension into a 10 ml round bottom centrifuge tube.
8. Cryogenic centrifugation: Centrifuge at 200 g for 3 minutes to remove the supernatant.
9. Removal of residual enzyme solution: Wash the precipitated protoplasts 3 times with 8% (w/v) mannitol. The amount of mannitol added depends on the number of protoplasts.
10. Cell suspension quality control: Resuspend the protoplasts in 8% (w/v) mannitol and determine the number of protoplasts and cell viability after cell digestion by acridine orange-propidium iodide (AO/PI) staining using a Countstar automated fluorescence cell analyzer.
11. Single-cell capture: Resuspend the quality-control-qualified protoplast samples to 800~1000 cells/μL and capture the protoplasts using a single-cell capture platform or a Liebing PanoCell dual-channel microchip.
12. Cautions:

• Avoid dragging during scalpel chopping to prevent damaging the protoplasts.
• Perform the experimental steps after digestion with the enzyme solution on ice.
• Maintain a certain angle of inclination of the centrifuge tube during sieving, so that the liquid flows down the wall of the tube to reduce cell loss caused by liquid impact.
• Use a wide-mouthed tip for the removal of supernatant and operate gently to prevent damage to protoplasts.
• Monitor the enzymatic digestion of protoplasts using microscopic examination and quality control processes every hour. This experiment should be continued for 6 hours, and it is recommended to determine the best time for cell viability of rice inflorescence protoplast preparation is 2 hours.