The root tip regions (~2 cm in length from root tip) were cut, longitudinally, into 3 - 4 small strips and then processed for protoplast isolation. Here, the root strips were transferred immediately and gently into the prepared enzyme solution (0.1 moluL-1 KCl, 0.08 moluL-1 MES, 0.02 moluL-1 CaCl2, 1.5% cellulase R10 (Yakult, Japan), 1% Pectolyase (Yakult, Japan), 0.4 moluL-1 mannitol, and 0.1% BSA), and digested at 25°C, with shaking (40 rpm), in the dark for the indicated period. An equal volume of washing buffer (WB) solution (0.1 moluL-1 KCl, 0.08 moluL-1 MES, 0.4 moluL-1 mannitol and 0.1% BSA) was added in the enzyme solution and the mixture was then filtered using nylon mesh (200 mesh/inch). The flow-through was centrifuged at 200 x g for 2 min to pellet protoplasts. To evaluate protoplast yield, under different isolation conditions, protoplasts were re-suspended with suitable WB solution and counted with a hemocytometer. Protoplast viability was determined by trypan blue staining, with the ratio of viable cells being more than 85%. The concentration of protoplasts was adjusted to 700–1200 cells/μL and cells were then processed with the 10x Genomics Single Cell Protocol (CG00052, RevC). The libraries were constructed using 10× Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1(1000268). In brief, cell suspensions in a chip were loaded on a Chromium Controller (10xGenomics, Pleasanton, CA) to generate single-cell GEMs (gel beads in emulsion). The scRNA-seq libraries were then prepared, according to the manufacturer's protocol. Qualitative analysis of the DNA library was performed on an Agilent 2100 Bioanalyzer. The concentration of DNA library was measured by Qubit (Invitrogen).