Time-course RNA-seq analysis of rice flag leaves across senescence-associated developmental stages
Time-course RNA-seq analysis of rice flag leaves across senescence-associated developmental stages
Flag leaves were collected from wild-type rice plants (Oryza sativa L. cv. Dongjin) grown under field conditions at Kyung Hee University, Yongin, Republic of Korea. Samples were harvested at five reproductive stages spanning natural senescence progression: one week before heading, heading, one week after heading, three weeks after heading, and five weeks after heading. At each stage, flag leaves were sampled based on panicle developmental status to ensure consistent stage definition. Two independent biological replicates were collected for each stage. All samples were immediately frozen in liquid nitrogen after harvest and stored at -80°C until RNA extraction. Rice seeds (Oryza sativa L. cv. Dongjin) were surface-sterilized with 50% sodium hypochlorite and germinated on half-strength Murashige and Skoog medium for 7 days at 28/22°C (day/night) under continuous light. Seedlings were transferred to soil in a greenhouse for acclimation and subsequently transplanted to a paddy field at Kyung Hee University, Yongin, Republic of Korea, where plants were cultivated under natural field conditions with standard agronomic management. Total RNA was extracted from frozen rice flag-leaf tissue using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. RNA quality was assessed prior to library preparation, and only high-quality RNA samples were used for sequencing. Extracted RNA was used for transcriptome analysis of wild-type flag leaves collected at the five developmental stages. Strand-specific poly(A)+ RNA-seq libraries were constructed from high-quality total RNA using the TruSeq Stranded mRNA Library Preparation Kit (Illumina), following the manufacturer's protocol. Briefly, polyadenylated RNA was enriched from total RNA, fragmented, and reverse-transcribed to generate first- and second-strand cDNA. After end repair, adapter ligation, and PCR amplification, the final libraries were subjected to sequencing.