title: GSM8261202: Vertical SDX cells bio4; Populus trichocarpa; RNA-Seq
library_construction_protocol: The protoplast isolation of SDX mainly followed our previously established protocol (Lin et al., 2014). The stems of normal or tension/opposite woods were cut into 8-cm segments and then debarked. The cell wall of the debarked stems were digested with cellulose-macerozyme solution (0.25 M mannitol, 20mM MES, pH 5.7, 20 mM KCl, 10 mM CaCl2．2H2O, 0.1% BSA, 1.5% cellulase R10, 0.4% macerozyme R10) for 3 hours at room temperature. After cell wall digestion, the stems were transferred into the modified MMG solution (4 mM MES, 0.25 M mannitol, 20 mM KCl) with shaking for 1 min to release SDX protoplasts. The SDX protoplasts were filtered through a 70-μm cell strainer and then centrifuged at 900g for 3 min at room temperature, and the filtered protoplasts were resuspended with the modified MMG solution. These protoplasts were further sorted. For cell sorting, a solution of FDA (fluorescein diacetate) at a final concentration of 0.008 mg/ml was used to stain live protoplasts. The staining process involved incubation for 5 minutes at room temperature in the dark. Subsequently, the stained protoplasts were centrifuged at 900g for 3 minutes at room temperature and then resuspended in the modified MMG solution. The FDA-stained SDX protoplasts were filtered through a 40-μm cell strainer and sorted using the BD FACSAria™ III cell sorter. A 488 nm laser was employed to select the FDA-stained SDX protoplasts. The sorting process involved narrowing down the selection based on forward-scattered light-height (FSC-H) and side-scattered light-height (SSC-H), followed by selection based on fluorescein isothiocyanate-area (FITC-A) and side-scattered light-area (SSC-A). Single-cell sequencing libraries of four biological replicates for vertical, tension, and opposite stem differentiating xylem (SDX) in P. trichocarpa were generated using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10X Genomics). The number of cells loaded following to the instructions in the 10x Genomics manual aimed at minimizing the formation of doublets or multiplets.