Direct mRNA Isolation

mRNA sequencing was carried out at the phenological stage BBCH65 (5th inflorescence) before and after 48 h of treatment application. mRNA was directly isolated using the Dynabeads mRNA Direct Micro Kit (Thermo Fisher Scientific, Carlsbad, CA, United States) following the protocol for mRNA isolation from tissues. Briefly, we ground 30 mg of frozen leaf samples for 3 min with the Tissue Lyser (Qiagen, Germany) together with 100 μl of lysis-binding buffer. The lysates were then combined with 20 μl of pre-washed Dynabeads Oligo (dT) and mixed by pipetting up and down three times. The sample tubes were placed in a mixer for 5 min to allow the mRNA to anneal to the Dynabeads Oligo (dT) and successively placed in DynaMag-2 Magnet (Thermo Fisher Scientific) for 1 min to discard the supernatant. Samples were then removed from the magnet and the Dynabeads-mRNA complex was resuspended in 100 μl of Washing Buffer A. Again, the supernatant was removed by placing the sample tubes in the DynaMag-2 Magnet and this step was repeated. 100 μl of Washing Buffer B was added to the remaining Dynabeads-mRNA complex and washed two times by discarding the supernatant using the DynaMag-2 Magnet. Finally, the Dynabeads-mRNA complex was eluted in 20 μl of ice-cold 10 mM Tris–HCl and incubated at 65–80°C for 2 min. Once the tubes were placed in the magnetic rack, we transferred the supernatant containing the purified mRNA into a new tube and its quality and quantity were checked by Agilent TapeStation 1500 (Agilent Technologies Inc., Santa Clara, CA, United States). The average mRNA yields obtained were 2,150 ± 479 pg/μl. Once extracted, the quantification method showed contamination from 18S or 28S sequences that were removed to avoid unwanted amplicons by performing an additional washing step at the end of the mRNA extraction protocol.