Sybr green dye

Total RNA was isolated from cells and tissues using Qiazol (Qiagen, Hilden, Germany) and complementary DNA synthesized by random hexamer priming with the Verso cDNA kit (Thermo Fisher, Waltham, MA, USA). quantitative real-time PCR was performed using Taq-Pro DNA polymerase (Denville Scientific, Holliston, MA, USA) and SYBR green dye (Lonza, Portsmouth, NH, USA). Fold changes were calculated by the ΔΔC_t method using B-actin as a standard, and normalized to the experimental WT control. Primer sequences are as follows: B-actin F: 5ʹ-AGCTTCTTTGCAGCTCCTTCGTTG R: TTCTGACCCATTCCCACCATCACA-3ʹ; CD36; F: 5ʹ-GAGCAACTGGTGGATGGTTT R: GCAGAATCAAGGGAGAGCAC-3ʹ; CPT1β F: 5ʹ-TTGCCCTACAGCTGGCTCATTTCC R: GCACCCAGATGATTGGGATACTGT-3ʹ; LCAD F:5ʹ-TCTTTTCCTCGGAGCATGACA R: GACCTCTCTACTCACTTCTCCAG-3ʹ; VLCAD F:5ʹ-CTACTGTGCTTCAGGGACACC R: CAAAGGACTTCGATTCTGCCC-3ʹ; PGC1α F: 5ʹ-AGCCGTGACCACTGACAACGAG R: GCTGCATGGTTCTGAGTGCTAAG-3ʹ; Leptin F: 5ʹ-TGAAGCCCAGGAATGAAGTC R: TCAAGACCATTGTCACCAGG-3ʹ; PPARα F: 5ʹ-TGTTTGTGGCTGCTATAATTTGC; R: GCAACTTCTCAATGTAGCCTATGTTT-3ʹ; Acox1 F: 5ʹ-CCTGATTCAGCAAGGTAGGG R: TCGCAGACCCTGAAGAAATC-3ʹ.

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).

Pollen ploidy in the jas-3 mutants was analyzed by collecting open flowers from individual plants into Eppendorf tubes, vortexing in 2 mL of 100-mM sodium phosphate buffer (pH 7) for 3 min, and filtering through a 50-µm nylon mesh. Pollen populations are characterized by an elevated high angle scatter (SSC) and autofluorescence, which allows haploid (1n) and diploid (2n) pollen to be discriminated, as previously described (Erilova et al., 2009 (link); Storme and Geelen, 2011 (link)). These two populations were gated and quantified (Supplemental Figure S2). For ploidy analysis of nuclei, leaf tissue was chopped in 2 mL of Galbraith buffer (45-mM MgCl₂, 20-mM MOPS, 30-mM sodium citrate, 1% (v/v) Triton X-100, pH 7.0) using a razor blade, filtered through a 50-µm mesh, stained with SYBR Green dye (Lonza), and analyzed on a CyFlow Space flow cytometer (Sysmex).