mRNA sequencing of WT rice plants and OsRIP1-overexpressing plants after methyl jasmonate treatment

Transcriptional profiling via mRNA sequencing was utilized to assess alterations in gene expression within rice plants following MeJA treatment. The objective of this study was to assess whether the overexpression of the ribosome-inactivating protein OsRIP1 (LOC_Os01g06740) affects the jasmonate signaling pathway in rice.Seeds of wild type (WT) rice Oryza sativa cv. Nipponbare and plants over-expressing OsRIP1 (LOC_Os01g06740) (T4 generation) from line J were used for plant materials.Plant growth Sterilized seeds were germinated in solid MS medium (pH 5.8) supplemented with 30 g/l sucrose, 8 g/l Agarose SPI (Duchefa Biochemie, Netherlands) and 1.12 mg/l Gamborg B5 vitamins (Duchefa) in square Petri dishes, sealed with micropore tape. Seeds of transgenic plants from line J were germinated on selective medium containing 4 mg/l phosphinothricin (Duchefa). Petri dishes were wrapped with aluminum foil and incubated in the dark in a plant chamber at 28degrees. After 4 days, the aluminum foil was removed, and germinated seeds were grown at 28degrees with a 16-h light/8-h dark cycle for an additional 3 days. One-week-old rice seedlings were grown hydroponically in the 1/2 Hoagland solution under the same conditions in a plant cabinet. The 1/2 Hoagland solution was refreshed daily.MeJA treatment 14-day-old plants were subjected to MeJA treatment. MeJA (Sigma-Aldrich, Darmstadt, Germany) was dissolved in absolute ethanol (Sigma-Aldrich) to obtain a 100 mM stock solution, and then added to the 1/2 Hoagland solution to reach the working concentration of 100 uM. Control seedlings were kept in the 1/2 Hoagland solution with 0.1% (v/v) ethanol. Shoots and roots were sampled at 3 h and 24 h, and stored at -80degrees. All treatments were set up for biological triplicates.mRNA sequencing Four treatment groups were set up for each indicated timepoint (3 h and 48 h), namely mock-treated WT plants, MeJA-treated WT plants, mock-treated T4 OsRIP1-OE transgenic rice plants line J, and MeJA-treated T4 OsRIP1-OE transgenic rice plants line J. Three independent biological replicates were performed for each treatment, containing 10-12 individual plants per replicate. Total RNA was extracted from freshly ground material using the Spectrum Plant Total RNA kit (Sigma-Aldrich). Shoot and root samples were analyzed separately. RNA integrity was checked using the Agilent BioAnalyzer 2100 (Agilent). Approximately 1 ug of RNA was used for 3' mRNA-Seq library construction using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina. To minimize lane effects, the samples were multiplexed, using the multiplexing sequencing adaptors provided in the Multiplexing Sample Preparation Oligo Kit (Illumina). Size selection was performed on a 2% agarose gel (Low Range Ultra Agarose, Biorad 161-3107). The denatured library was diluted to a final concentration of 6 pM and loaded on a flow cell (Illumina). After cluster generation, the multiplexed library was sequenced on an Illumina NextSeq500 (75 cycles, single end, high output).