design: scRNA-seq of mature Arabidopsis thaliana leaf
library_construction_protocol: For isolation of leaf protoplasts (~5 excised leaves from four-week-old seedlings pooled per sample), a “tape-sandwich” method was used, where the abaxial epidermis of leaves was peeled using adhesive tape (Scotch® Magic™, 3M) and leaves were immediately immersed in a cell-wall degrading enzymatic buffer in protoplasting buffer (0.6 M mannitol, 20 mM KCl, 10 mM CaCl2, 20 mM MES, 0.1% BSA, 1.0% cellulase R10, 0.3% macerozyme) at pH 5.7. The enzymatic reaction was performed for 1 h at room temperature with gentle rotation (30 rpm) in the dark. The solution was filtered through pre-wet 70 μm nylon mesh, collected by centrifugation at 200 × g for 6 min and resuspended in wash buffer (0.6 M mannitol, 20 mM KCl, 10 mM CaCl2, 20 mM MES). Arabidopsis (Arabidopsis thaliana L. Heynh.) cv. Columbia-0 (Col-0) was grown in a controlled-environment growth chamber (Weiss Technik).  Beads in emulsion (GEM) generation and droplet barcoding were performed according to the 10x Genomics protocols with Chromium Next GEM Single Cell 3'Kit v3.1 and Chromium Next GEM Chip G Single Cell Kit, aiming for 10,000 cells per sample. The droplets were further processed for short-read sequencing using the 10x Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) per the manufacturer's protocol (10x user guide CG000315).