Acid washed beads

To extract RNA, 50 ml mid-log phase yeast cultures were pelleted and resupended in 1 ml Trizol (Thermo Fisher Scientific), and then 400 µl acid-washed beads (Sigma-Aldrich) were added. Tubes were sequentially vortexed five times for 20 s with 1-min intervals. 150 µl chloroform was added, and the samples were mixed. The tubes were centrifuged in a microfuge for 15 min at 12, 000 × g. The aqueous layer was collected, and 350 µl isopropanol was added. The resulting precipitate was collected via centrifugation in a microfuge for 15 min at 12,000 × g and washed in 75% ethanol. The resulting pellet was resuspended in 20 µl of nuclease-free H₂O. qRT-PCR was performed using 300 ng RNA with the CFx Connect Real-Time system with the iTaq Universal SYBR Green One Step Kit (Bio-Rad) according to the manufacturer’s instructions. Primers were designed to amplify a 200-nt region just upstream of the STOP codon. Samples were run in triplicates and normalized to ACT1 mRNA, and the fold change was calculated using 2^−ΔCt for each tested RNA.

For each replicate, approximately 100 mg of frozen sample (stored at -80°C) of SWE-treated/untreated roots and leaves were used. Samples were weighted in 2 mL tubes containing acid-washed beads (Sigma Aldrich, Australia) and kept frozen in liquid nitrogen before being homogenized at 5 m/s × 30 s × 3 times in a FastPrep-24™ instrument (MP Biomedicals, Australia). A volume of 500 μL extraction solution containing a 2:3:3 ratio of water: acetonitrile: isopropanol (Sigma Aldrich, Australia) was added to the 2 mL tubes and again homogenized (5 m/s × 30 s × 3 times). Samples were then subjected to further centrifugation (13,300 g × 5 min) and the supernatants were then diluted to a 1:1 ratio with Milli-Q water. Finally, 150 µL of the diluted supernatant was transferred to 2 mL vials (Thermo Fisher Scientific, Australia) for the UHPLC-MS analysis. Two blank samples and two pooled biological quality control samples (PBQC) were prepared. The two blank samples contained only extraction solution and were used for the detection and identification of background compounds, which were subsequently removed. The two PBQC samples were created by mixing equally all individual samples together, and it was analyzed at the beginning and the end of the UHPLC-MS measurement (Supplementary Figure S1).

We adapted previously published 3C methods^{62 (link),63 (link)}. In all, 2 × 10⁹ cells in 200 ml SCD were fixed in 3% formaldehyde (Sigma) for 20 min at 25 °C, and then quenched with 35.28 ml of 2M glycine for 20 min at room temperature. Cells were recovered by centrifugation and washed with the same medium. The cell pellet was resuspended in 1 ml TBS, 1% Triton-X and 1X protease inhibitor cocktail (Thermo Scientific). Cell lysis was obtained by adding 500 μl acid-washed beads (Sigma) and vortexing for 5 min, 5 times with 2-min break on ice. The chromatin was recovered through centrifugation, washed with 1 ml TBS, resuspended in 500 ul 10 mM Tris buffer and digested with DpnII (NEB) overnight at 37 °C. The digested DNA fragments were ligated by T4 DNA ligase (Thermo Scientific) for 4 h at 16 °C. Crosslink was reversed by incubation with proteinase K at 65 °C overnight. DNA was purified through a phenol/chloroform extraction. RNA was removed through an RNase A treatment, and DNA was purified again by DNA clean and concentrator-5 Kit (Zymo). Primers were designed for the regions of interest near DpnII cutting sites (Supplementary Table 2), and PCR reactions were performed for testing interactions between these regions.