GSM8819790: star petal protoplasts; Petunia x hybrida; RNA-Seq
Protoplast isolation was performed in sterile conditions. WT, star and wico corollas (ca. 15, 30 and 30 flowers per replicate, respectively) were briefly soaked in 70% ethanol and in 0.5% bleach for 30'' then rinsed 3 times with sterile water. Sterile corollas were transferred in a petri dish containing 2 mL of Digestion Mix (0.4% macerozyme R-10, 0.8% Cellulase Onozuka R-10, w/v in TEX Buffer (3.1 g/L Gamborg B5 salts, 500 mg/L MES, 750 mg/L CaCl2*2H2O, 250 mg/L NH4NO3, 136.9 g/L Sucrose, pH 5.7)) and cut in ca. 0.5 cm² pieces, using a new scalpel blade for each corolla to reduce tissue wounding. 10 mL of Digestion Mix were added and digestion was performed for 5 h at 26°C in the dark, with gentle orbital agitation (20 rpm) for the last 15'. After filtration through a 40 µm mesh, the volume was adjusted to 25 mL using 0.4 M Sucrose (492 mOsm.kg-1 H2O) and the mixture was centrifuged for 10' at 100 g with acceleration 2/9 and deceleration 0/9 with a swing-out rotor. The underlying buffer was removed using a peristaltic pump (Gilson MINIPULS™ Evolution) connected to a sterile Pasteur pipette without perturbing the protoplast layer, at a rate of ca. 100 µL/sec. After adjusting again the volume to 25 mL using 0.4 M Sucrose, the whole process was repeated twice. Protoplast concentration and viability was assessed using a Kova slide and staining with 1% Evans Blue dye solution (w/v in 0.4 M Sucrose). Protoplast suspensions were adjusted to 345, 480, 590 and 560 cells/µL with Mannitol-BSA (0.44 M Mannitol, 0.1 % BSA (w/v), 498 mOsm.kg-1 H2O) for WT (first replicate), star, wico and WT (second replicate) respectively. Protoplasts were loaded in the 10x Genomics Chromium chip and libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) kit following manufacturer's instructions.