Homogeneous low nitrate, control, biol. Repl.2, paired 1
For this transcriptome study seeds of the maize inbred line B73 were surface sterilized and germinated in moist germination paper. About 7-day old uniform seedlings were transferred to hydroponics system cultured by low nitrate (0.5 mM) nutrient solution. The seedlings were cultured in this system until the 7th whorl shoot-borne roots initiate, during this culture time, the nutrient solution was replaced every three days and aerated continuously. When the shoot-borne roots grow to 7 cm length then the roots were split-treated by low nitrate (0.5 mM) or high nitrate (4 mM) solution. After 24 h of incubation in these split-root system (Yu et al. 2014), shoot-borne roots tips (5 mm length) were dissected and the root stele tissue (5-25 mm from root tip) was mechanically separated from cortex according to Saleem et al. 2010. Experiments were performed in four biological replicates each consisting of 15 pooled root steles resulting in 8 distinct samples (2 treatments x 1 tissue x 4 replicates). Pooled shoot-borne root steles were ground in liquid nitrogen, and total RNA was isolated with the RNeasy Plus Universal Mini Kit (Qiagen, Venlo, Netherlands). RNA quality was assessed via agarose gel electrophoresis and a Bioanalyzer (Agilent RNA 6000 Nano Chip, Agilent Technologies, Santa Clara, USA). For all samples, a RIN ≥ 9.7 detected. cDNA libraries for Illumina sequencing were constructed according to the protocol of the manufacturer (TruSeq RNA Sample Preparation, Illumina, San Diego CA, USA). For sequencing, each library was indexed by an Illumina TruSeq Adapter in which no adapter was used more than twice. Indexed libraries were loaded onto a flow cell following an incomplete block design with four pooled libraries per lane. Cluster preparation and pair-end read sequencing were performed according to the manufacturer’s instructions (HiSeq 2000, Illumina, San Diego CA, USA).