title: Illumina NovaSeq 6000 paired end sequencing; Early transcriptomic responses of rice leaves to herbivory by Spodoptera frugiperda
design: Early transcriptomic responses of rice leaves to herbivory by Spodoptera frugiperda
library_construction_protocol: After treatment, the plants were left for 2 hours in the culture chamber. Then the leaves from five seedlings were pooled, and immediately frozen in liquid nitrogen before being stored at -80°C.  Oryza sativa, japonica cv Kitaake wild-type (WT) rice seeds were used for the experiments. Seed coats were removed, and seeds were sterilized in 70 % ethanol for 1 min, then in 3.7 % bleach for 30 min. They were rinsed six times with sterile water and sown on half‐strength Murashige & Skoog (½MS) medium including Gamborg B5 vitamins (Duchefa Biochemie BV, Haarlem, the Netherlands) supplied with 0.5 % plant agar (Duchefa), pH 5.8. After two days in a culture chamber (27°C, 70 % relative humidity, 12 hours photoperiod, 12,000 lux light intensity), germinated seeds were vertically placed in a square Petri plate contain ½MS + B5 medium supplied with 0.7 % plant agar in its one third inferior part. Five seeds were placed per plate and grown for four days under the same conditions.  Total RNA from rice leaves was extracted with the RNeasy Plant Mini kit (Qiagen) with an additional DNase treatment step to remove genomic DNA contamination. The concentration and purity of the RNA samples were measured with a NanoDrop spectrophotometer. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.