Mo17xB73, drought treatment by PEG, biological replicate 1
Seeds of the inbred lines B73 and Mo17 and the two reciprocal hybrids B73xMo17 and Mo17xB73 were germinated in paper rolls in distilled water as previously described (Hoecker et al., 2006). Four to 5 days (16 h light, 28 °C; 8 h dark, 21°C) after germination seedlings with a primary root length of 2 to 4 cm were transferred to new paper rolls soaked with polyethylene glycol (PEG8000 Mr 7,300-9,000; Roth, Karlsruhe, Germany) solutions with a water potential of  0.8 MPa or distilled water. Exact water potentials of the PEG8000 solution were determined as previously described (Opitz et al., 2014). Paper rolls with seedlings were incubated in aerated PEG solutions or distilled water for 6h (24°C, 24 h included 8 h darkness, 18 °C). Before and after treatment, seedlings were photographed and primary root length was measured using WinRHIZO (http://www.regent.qc.ca/). Whole primary roots were harvested, immediately frozen in liquid nitrogen and stored at  80 °C until RNA isolation. Experiments were performed in four biological replicates each consisting of 10 pooled roots.  The pooled primary roots tissues were ground under liquid nitrogen and RNA was isolated as previously described (Winz and Baldwin, 2001). RNA quality was assayed via agarose gel electrophoresis and a Bioanalyzer (Agilent Technologies, Boeblingen, Germany). Only samples with an RIN (RNA integrity number; Schroeder et al., 2006) ≥ 9,1 were used for downstream analyses. The cDNA libraries for Illumina sequencing were constructed in accordance with the protocol of the manufacturer (TruSeq RNA Sample Preparation, Illumina, San Diego CA, USA). For sequencing, four libraries were pooled in one lane of a flow cell. Each library per lane was indexed by one of the adapters AR001, AR008, AR010, or AR011 (Table S1). The indexed libraries were loaded onto a flow cell according to an incomplete block design (generated with CycDesigN, http://www.cycdesign.co.nz/; Figure S1). Cluster preparation and single read sequencing were performed according to the manufacturer’s instructions (HiSeq 2000, Illumina, San Diego CA, USA).
University of Bonn
Mo17xB73-drought-1