Gel purification kit

The phylogenetically informative V3-V4 region of the 16S rRNA (rRNA) gene was amplified using universal primer 347F/803R (66 (link), 67 (link)). The dual-barcoding approach was applied as previously described (68 (link), 69 (link)) to label the 16S rRNA gene amplicons of each sample. Briefly, the 6-mer barcodes were attached on the 5′ ends of both forward and reverse PCR primers such that the 16S rRNA gene PCR amplicons from each sample contained a unique dual-barcode combination. The PCR primers were synthesized by Sangon Biotech, Shanghai, China, and the primer sequences are shown in Table S1 in the supplemental material. The 25-μl PCR mixture contained 300 ng of sample DNA as a PCR template, 1 μl of 10 μM forward and reverse 16S primers, and 12.5 μl of 2× HotMaster Taq DNA mix (Tiangen Biotech, Beijing, China). The PCR was performed on an Applied Biosystems model 2720 thermal cycler (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 94°C for 3 min and then at 94°C for 30 s, 58°C for 30 s, and 72°C for 20 s for 30 cycles followed by 72°C for 4 min. The integrity of the PCR products was verified by agarose gel electrophoresis. After a purification step was performed with a gel purification kit (Promega, Madison, WI, USA), the 16S PCR amplicons were pooled at equal levels of molarity, freeze-dried, and submitted to the New York Genome Center for sequencing.

3.5 dpg ESTpro::MUTE seedlings were submerged in 2 uM estrodial or ½ liquid MS as a control for 4 hours. Then 0.5 g of seedlings were harvest and ground in liquid nitrogen. Nuclei were prepared as described previously ^{38 (link)} with the following modifications. The isolated nuclei were washed twice in 1 ml of HBB buffer (25 mM Tris-Cl pH 7.6, 0.44 M sucrose, 10 mM MgCl2, 0.1% Triton-X, 10 mM beta-mercaptoethanol). Nuclei were then treated with 0.5 U/ul final concentration of Micrococcal Nuclease (NEB) in digestion buffer (16 mM Tris-Cl, pH 7.6, 50 mM NaCl, 2.5 mM CaCl2, 0.01 mM PMSF and EDTA-free protease inhibitor cocktail (Roche)) for 3 min at 37°C. Digestion was stopped with 10 mM EDTA and treated with Proteinase K (Roche). DNA was purified with phenol:chlorophorm:isoamyl alcohol (25:24:1) extraction and precipitated with 1/10 volume of 5 M sodium acetate and 2 volumes of ethanol. Purified DNA was run on 2% agarose gel and bands corresponding to ~150 bp were cut and purified with a Gel Purification kit (Promega). Libraries were constructed using the Nugen Ovation Ultralow Library System V2 following the manufacturer’s protocol. 75 bp of pair-end reads were sequenced on NextSeq 4000.