Isolation and Purification of Pollen Nuclei for Genomic Analysis

Pollen nuclei of wild-type (Col-0) and mutant dme/+, ros1, and dme/+;ros1 plants were purified by FACS using SYBR Green staining as previously described^{49 (link),50 (link)}. Briefly, open flowers were collected into a 50 mL falcon tubes and vortexed in 10 mL of Galbraith buffer (45 mM MgCl₂, 30 mM Sodium Citrate, 20 mM MOPS, 1% Triton-100 pH 7.0) for 3 min, at room temperature. This crude fraction was then filtered through Miracloth (Calbiochem) and centrifuged for 1 min at 2600 × g to concentrate the pollen fraction. The pollen was then transferred to a 1.5 mL Eppendorf tube containing ~100 μL of acid-washed glass beads (425–600 μm, Sigma) and vortexed continuously at maximum speed for 3 min, to break the pollen cell wall. The fraction containing the released nuclei was then filtered through a 10 μm mesh (Celltrics, Sysmex-Partec) to exclude pollen debris and stained with SYBR Green dye (Lonza). FACS was performed using a FACSAria IIU cell sorter (BD Biosciences), using the integrated FACSDiva v6.1 software with 70 µ nozzle at 70 psi. A 488 nm laser was used for SYBR Green excitation, which was detected by a 530/30 nm band-pass filter (Supplementary Fig. 2). Approximately 500,000 nuclei from each genotype and cell type were purified, and genomic DNA was extracted using the Masterpure kit (Epicenter).