title: E21-R2
organism: Sorghum bicolor
description: Protocols: we carefully collected seed samples every day immediately following successful pollination. While we collected five replicates for samples obtained at 5, 10, 20, 25, and 30 dpa to facilitate metabolomic analysis, three biological replicates were collected for all other samples. For each replicate, 10 uniform panicles were selected, within plots that extended 20 feet in length, each containing 50 plants. Within these plots, we meticulously identified and labeled the primary uniform tillers and their corresponding spikes, which were utilized for subsequent sample collection. The middle part of each sampled panicle was used for seed collection to avoid samples from different flowering time and seed developmental status. All samples were collected during the morning hours (between 9:00 AM to 11:00 AM) to minimize potential circadian influences on the transcriptome and metabolome. After collection from the field, the spikes were immediately transported to the laboratory and dissected on ice, using scalpels and tweezers, to isolate embryos and endosperm. These samples were then promptly snap-frozen in liquid nitrogen. After collection from the field, the spikes were immediately transported to the laboratory and dissected on ice, using scalpels and tweezers, to isolate embryos and endosperm. The study used Sorghum bicolor L., cultivar BTx623, in a field condition at the Quaker Research Farm, located in Lubbock, TX at coordinates 33°35'52.9\"N 101°54'21.4\"W, and an elevation of 992 meters above sea level, during the summer of 2022. The experimental design employed was a randomized complete block design with three replications for RNA–seq sample and five replicates for metabolome analysis. The site experiences a semi-arid climate characterized by an average yearly precipitation of 469 mm, primarily occurring from May to October. The annual average high and low temperatures are 23.3°C and 7.8°C, respectively. The soil type of the experimental location is identified as Amarillo sandy clay loam, specifically categorized as fine-loamy, mixed, super active, thermic Aridic Paleustoll The isolation of total RNA was accomplished using TRIzol reagent. The quality of extracted RNA samples was examined by agarose gel electrophoresis. Standard protocols for the Illumina NovaSeq platform were used for construction of RNA-seq libraries.