30 m mesh

Leaves from four-week-old plants were fixed in 3.7% formaldehyde in cold Tris buffer (10 mM Tris-HCl pH 7.5, 10 mM NaEDTA, 100 mM NaCl) for 20 minutes. Formaldehyde solution was removed, and leaves were washed twice for 10 minutes in Tris buffer. The leaves were then finely chopped with razor blade in 500 μl LB01 buffer (15 mM Tris-HCl pH7.5, 2 mM NaEDTA, 0.5 mM spermine-4HCl, 80 mM KCl, 20 mM NaCl and 0.1% Triton X-100). The lysate was filtered through a 30 µm mesh (Sysmex Partec, Gorlitz, Germany). 5 μl of lysate was added to 10 μl of sorting buffer (100 mM Tris-HCl pH 7.5, 50 mM KCl, 2mM MgCl2, 0.05% Tween-20 and 5% sucrose) and spread onto a coverslip until dried. Cold methanol was added onto each coverslip for 3 min, then rehydrated with TBS-Tx (20 mM Tris pH 7.5, 100 mM NaCl, 0.1% Triton X-100) for 5 min. The coverslips were mounted onto slides with Vectashield mounting medium DAPI (Vector Laboratories, Burlingame, CA). Nuclei were imaged on a Nikon Eclipse Ni-E microscope with a 100X CFI PlanApo Lamda objective (Nikon, Minato City, Tokyo, Japan). Digital images were obtained using an Andor Clara camera. Z-series optical sections of each nucleus were obtained at 0.3 μm steps. Images were deconvolved by imageJ using the deconvolution plugin.

Rosette leaves from three-week-old plants were finely chopped in 0.5 ml Galbraith buffer (45 mM MgCl2, 20 mM MOPS, 30 mM sodium citrate, 0.1% Triton X-100, 40 μg/μl RNase A) using a razor blade. The lysate was filtered through a 30 µm mesh (Sysmex Partec). Propidium iodide (Sigma, St. Louis, MO) was added to each sample to a concentration of 20 µg/ml and vortexed for 3 seconds.
Each sample was analyzed using a BD FACS LSR Fortessa X20 (Becton Dickinson, Franklin Lakes, NJ). Quantification (nuclei counts and robust CV values) was performed using Flowjo 10.0.6 (Tree Star, Ashland, Oregon).