Circadian regulation of transcriptome in spinach leaves under varying nitrogen levels (aligned to Sp75 reference genome)

The goal of this study was to compare differentially expressed transcripts in spinach leaves under contrasting nitrogen levels throughout the day and night using transcriptome profiling (RNA-seq) Method: 48-day-old spinach leaf samples (cultivar- Space) were collected from 3 independent plants grown under contrasting levels of nitrogen (high: HN, low: LN) during four different times of the day (T1- End of the dark cycle; T2 – Middle of the light cycle; T3 – End of the light cycle; T4 – Middle of the dark cycle) and were flash-frozen in liquid nitrogen. The homogenized samples were used for total RNA extractions. The T1 set of samples from 3 independent plants under high nitrogen were labeled as (HNT1_1, HNT1_2, HNT1_3) while under low nitrogen as (LNT1_1, LNT1_2, LNT1_3). Similarly, the samples from the remaining 3-time points under both nitrogen levels were labeled. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand of cDNA was synthesized using random hexamer primers, followed by the second strand of cDNA synthesis using dUTP for directional library. Quantified libraries were pooled and sequenced on NovaSeq X Plus Series, according to effective library concentration and data amount.