organism: Flaveria bidentis
description: Protocols: For single-protoplast isolation, leaves from three different plants were harvested and finely chopped in a 9 cm Petri dish containing 15 mL of freshly prepared enzyme cocktail (containing cellulase RS, macerozyme, mannitol, KCl, MES, CaCl₂, and BSA). Digestion was performed for 2.5 hours in the dark at 25°C on a shaking incubator (40 rpm) and stopped by adding an equal volume of W5 buffer. Released protoplasts were filtered twice through a 40 µm strainer, collected in a 50 mL tube, centrifuged at 100 × g for 5 minutes, and washed and resuspended three times for purification. After the final wash, the supernatant was removed, leaving 0.5–1 mL of protoplast suspension in 0.4 M mannitol. Protoplast integrity and concentration were assessed using a hemocytometer under a light microscope. High-quality suspensions were adjusted to ~1000 protoplasts/µL. For single-nuclei sequencing, leaves from the same plants were harvested and finely chopped in a 5 cm Petri dish containing 1.5 mL of freshly prepared nuclei purification buffer, containing 1 U/µL Protector RNase Inhibitor. The chopped sample was filtered sequentially through 70 µm and 40 µm strainers. Nuclei were stained with Hoechst and sorted via FACS. Sorted nuclei were collected in a 1.5 mL tube containing BSA and Protector RNase Inhibitor, then counted using a hemocytometer. Nuclei suspensions were adjusted to ~400–600 nuclei/µL. Gynandropsis gynandra seeds were germinated on moist filter paper in the dark at 32°C for 32 hours, then sown directly onto soil.  Flaveria bidentis seeds were placed on ½-strength Murashige and Skoog medium in 0.8% (w/v) agar (pH 5.8) and grown for 10 days in a growth chamber at 24°C, photoperiod 16 hours light and 8 hours dark before being transferred to soil.  After sowing G. gynandra and transplanting F. bidentis, plants were maintained in a growth cabinet under a 16-hour light/8-hour dark cycle at 24°C, with 60% relative humidity, and a photon flux density of 400 μmol m−2 s−1. Plants were grown for 6 weeks until leaves were harvested for single-protoplast or single-nuclei RNA sequencing. A total of 10,000 protoplasts (targeting 6,000 recovered cells) or 16,000 nuclei (targeting 10,000 recovered nuclei) were loaded per well according to the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 protocol for GEM Generation, Barcoding and cDNA amplification.  Single-protoplast and single-nuclei libraries were constructed using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) according to the manufacturer's standard protocol.