Time-course RNA-seq of flag leaf node during senescence in field-grown Triticum aestivum cv. Paragon under two nitrogen fertilisation levels
Time-course RNA-seq of flag leaf node during senescence in field-grown Triticum aestivum cv. Paragon under two nitrogen fertilisation levels
Flag leaf nodes were carefully dissected and immediately frozen in liquid nitrogen to preserve RNA integrity. Each biological replicate consisted of 20 flag leaf nodes collected from plants randomly selected within each plot. Stems had been labelled at anthesis to track developmental timing. Sampling was conducted at eight distinct time points after anthesis, chosen based on the progression of senescence. The selected time points for each nitrogen treatment were as follows: N100 – 0, 7, 14, 21, 25, 28, 31, and 34 days post-anthesis (DPA); N200 – 0, 7, 14, 21, 28, 32, 35, and 38 DPA. Samples were stored in -80 oC until RNA extraction. Triticum aestivum cv. Paragon was grown in a field trial under two nitrogen fertilisation levels: 100 and 200 kg N ha⁻¹, with three replicate plots per treatment. Each plot was 2.75 m × 9 m. The trial was part of the Defra-funded Wheat Genetic Improvement Network (WGIN) Diversity 2016 field experiment (https://grassroots.tools/fieldtrial/study/60827ff102700f28ce3c1198). The experiment was conducted during the 2015/2016 growing season at Blackhorse Field (coordinates: 51.804819, -0.398773), located at Rothamsted Research Experimental Farm in Harpenden, UK. Standard farm practices were followed for herbicide, fungicide, and insecticide applications. The frozen plant material was ground using a SPEX SamplePrep 6870 freezer mill (SPEX SamplePrep). The total RNA isolation was conducted according to Verwoerd et al. (1989) with the modifications described in Buchner and Hawkesford (2014). To ensure no DNA carryover, DNase treatment was performed using RQ1 RNase-Free DNase (Promega). After DNase treatment RNA was further purified by phenol/chloroform/isoamyl-alcohol purification. Prior the sample submission, the extracted RNA was further purified with Plant RNeasy kit (Qiagen) following the provided RNA clean-up protocol. RNA concentration was determined using the Qubit Broad Range Assay (Invitrogen), and purity was assessed via A260/A280 ratio using a NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA integrity was also evaluated by electrophoresis of 500 ng RNA on a 1% agarose TAE gel.  Quality control, poly-A selection for rRNA removal, library preparation, and multiplexing were performed by the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics, Oxford University, according to their established standard procedures. RNA library preparation utilized the Illumina TruSeq Stranded mRNA kit.