title: NextSeq 500 sequencing; Transcription profiling of Populus x canescens leaves to evaluate the effect of treatment with different nitrogen forms and concentrations
design: Transcription profiling of Populus x canescens leaves to evaluate the effect of treatment with different nitrogen forms and concentrations
library_construction_protocol: Leaves were cut off and directly frozen in liquid nitrogen Populus x canescens plants were propagated and cultivated as described by Müller et al. (2013). Plants were in vitro multiplicated by micro cuttings and grown on half strength Murashige & Skoog medium for four weeks at 23 °C-24 °C, 60-85 μE×m−2×s−1 photosynthetically active radiation (PAR)  and a 16 h/8 h day/night regime. Subsequently, the plants were transferred to the greenhouse and further cultivated at 21 °C under ambient light with additional illumination of 150 µE×m−2×s−1 PAR 16 h/8 h day/night cycle. They were potted into 0.65 L pots containing a mixture of washed and twice autoclaved sand (0.71-1.21 mm and 0.4-0.8 mm particle size and soil a ratio of 10:2 v/v). For acclimation the plants were covered with a transparent plastic beaker for one week, which got gradually lifted during the second week and ultimately removed after 14 days. During two weeks of acclimatization phase all plants were treated with modified Long Ashton nutrient solution (Hewitt, 1952) containing 2 mM KNO3 excess nutrient solution ran off. Plants were divided into two groups and watered daily with modified Long Ashton nutrient solution, containing either ammonium (2 mM NH4Cl) or nitrate (2 mM KNO3) as nitrogen sources. The plants got transferred into 3 L pots containing the same substrate after 4 weeks of greenhouse cultivation. After eight weeks of ammonium or nitrate pre-treatment, each group was split into three subgroups and supplied with 0.4 mM, 2.0 mM or 8.0 mM NH4Cl or KNO3, respectively, for three weeks. Each of the six treatment groups consisted of 11 individual plants, which were randomized regularly. After completion of three week treatments, the leaves were harvested for transcriptome analysis.  RNA from Poplar leaves was extracted using either the innuPrep Plant RNA kit (Analytik Jena, Germany; 8 mL treatment groups) according to the manufacturer's instructions, or the CTAB extraction protocol (Chang et al., 1993; 0.4 and 2 mM treatment groups). Libraries were constructed using the TrueSeq RNA Library Prep Kit (Illumina) according to the manufacturer's instructions