RNAseq Library Construction and Analysis

For RNAseq library construction, rRNAs were removed from total RNAs with the RiboMinus Plant kit (ThermoFisher, Waltham, MA, USA) for mRNA enrichment. The mRNAs were degraded into 300-nt fragments with an RNA fragmentation reagent (Ambion, USA). cDNAs were synthesized by reverse transcription with random hexamer primers. RNAseq libraries were generated using an NEBNext Ultra RNA library prep kit (NEB, Ipswich, MA, USA), and 150-bp pair-end RNA sequencing was conducted using the Illumina NovaSeq 6000 platform at the Personalbio company. Reads of RNAseq data were mapped to the rice genome v7 and the PXO99A genome separately [54 (link)]. The significance of differentially expressed genes (DEGs) was determined by a fold change ≥ 2 and a p-value < 0.05.