Dnase 1 enzyme

Total RNA was extracted from plant tissues using TRIzol and the concentration was measured at 260 nm using a Nanodrop 2000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Equal amounts of total RNA were treated with DNase I enzyme (Roche, Basel, Switzerland) and converted into first-strand cDNA using SuperScript III (Invitrogen, Carlsbad, CA). The cDNA was then used to check target gene expression by real-time RT-PCR with gene-specific primers. Primer sequences are listed in Supplementary Data 1.

PBS-washed sperm cells were fixed with 4% paraformaldehyde and stored at 4 °C until treatment for the DNA fragmentation measurement. Sperm DNA fragmentation was measured using the TUNEL detection assay (Cell Death Detection Kit POD^®, Roche, France) as reported previously [29 (link)]. Briefly, aliquots of fixed sperm cells were washed in PBS, followed by permeabilization in 100 µL of a solution containing 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. Labeling was performed after washing with PBS containing 1% BSA (1000 g, 5 min). Counterstaining with propidium iodide (PI) allowed evaluation of sperm permeabilization. Negative controls were obtained by incubating sperm cells without enzyme (terminal deosynucleotidyl transferase [TdT]). Positive controls were performed by incubating sperm cells with Dnase I enzyme (3 UI, Roche, France). Analysis was performed on a minimum of 20,000 cells using a BD FACS Aria SORP cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Total RNA was extracted from plant tissues using TRIzol and the concentration was measured at 260 nm using a Nanodrop 2000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Equal amounts of total RNA were treated with DNase I enzyme (Roche, Basel, Switzerland) and converted into first-strand cDNA using SuperScript III (Invitrogen, Carlsbad, CA). The cDNA was then used to check target gene expression by real-time RT-PCR with gene-specific primers. Primer sequences are listed in Supplementary Data 1.