Meristematic zone, control, biol. Repl. 2
For this transcriptome study seeds of the maize inbred line B73 were surface sterilized and germinated in moist germination paper. Seedlings with a primary root length of 2 to 4 cm were transferred to new paper rolls soaked with polyethylene glycol (PEG8000) solutions with a water potential  0.8 MPa for water deficit, or distilled water for control experiments. After 6 h of incubation in aerated PEG solution or distilled water, primary roots were dissected into four distinct tissues. First, the apical 2 mm of the root were collected, containing root cap and meristematic zone. Second, the proximal zone adjacent to the root tip up to the part of the root where the root hairs became visible was sampled, which corresponds to the elongation zone. Finally, the distal differentiation zone, from the root hair zone to the coleorhiza, was mechanically separated into cortex and stele according to Saleem et al. 2010. Experiments were performed in four biological replicates each consisting of 10 pooled root tissues (of in total 40 seedlings) resulting in 32 distinct samples (2 treatments x 4 tissues x 4 replicates). Pooled primary root tissues 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 ≥8.5 was 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 single read sequencing were performed according to the manufacturer’s instructions (HiSeq 2000, Illumina, San Diego CA, USA).