Approximately 1000 roots per sample (wild type and p35S:MIR396, uncut controls and 5 days after cut) were collected per quadruplicate for protoplasting. The seeds were sown on nylon mesh, roots were cut at approximately 1cm from the tip, and placed in digesting solution (20mM MES, 400mM mannitol, 20mM KCl, 1.5% cellulase, 0.4% macerozyme, 10mM CaCl2) covered from the light. To enrich the samples with meristematic tissue, the roots were incubated for 20 minutes until the transition zone (between the proliferation and elongation zones) was digested and the tips were released, which was confirmed under microscope. Then, the excess tissue was filtered out using a 1 mm-pore mesh. Digestion continued for 90 minutes and, after filtering the solution twice with a 40µm pore cell strainer, cells were centrifuged down for two minutes at 300rcf and 4°C, washed with washing buffer (enzyme-free digestion solution) and centrifuged again with the same conditions. Buffer was discarded, leaving a small amount at the bottom to resuspend protoplasts, which were then counted in a Neubauer chamber under a light field microscope, and diluted to a concentration of 1000cells/µl. 12000 cells per replicate were sorted on a 10x Chromium platform and libraries were prepared using a 3' Gene Expression v3.1 kit, following the provider's instructions.