title: GSM7965619: Leaf, shr2, rep2, scRNAseq; Setaria viridis; RNA-Seq
library_construction_protocol: Leaf segments were sectioned in ∼3-mm fragments using a scalpel. Fragments were placed in a small petri dish and weighted to calculate protoplast yield. Protoplasting solution was added immediately after weighting, which consisted of 1.25% (w/v) Cellulase R-10, 2% (w/v) Cellulase RS, 0.4% (w/v) macerozyme R10, and 0.2% (w/v) pectolyase Y23. Tissue digestion was carried out with gentle shaking at room temperature for 1 hour and 40 minutes. The cell suspension was then filtered using a 40 μm nylon filter and cells were then centrifuged at 200 rcf for 5 min. Cells were resuspended in pre-filtered (0.22 µm) washing buffer (0.4 M mannitol, 0.02 M MES, 0.02 M KCl, 0.01 M CaCl2, 0.015 mM BSA). Cells were then re-suspended at a concentration of 2000-3000 cells/μL and loaded into a Single Cell A chip (10× Genomics). Approximately 10,000 cells were loaded per replicate. Three independent replicates per genotype were performed. Chips were loaded on a Chromium Controller (10x Genomics) to generate single-cell GEMs. Single-cell libraries were then prepared using the Chromium Single Cell 3´ library kit, following manufacturer instructions. The quality of resulting DNA libraries was assessed with an Agilent TapeStation system.