Sample 5
Fagus sylvatica
Protocols: Roots were quickly separated from soil, washed under running tap water, rinsed with deionized water, dried with paper towel, and immediately frozen in liquid nitrogen. Young (approx. 7 to 8 years) Fagus sylvatica L. trees were collected at a natural regeneration forest with intact roots with original forest soil and litter layer. Trees were kept outdoors under ambient conditions, protected from rain but regularly watered with demineralized water (to keep soil moisture content at approx. 0.32 m³ water per m³ soil) and a shading net to simulate natural forest light conditions. Trees were randomly relocated on a weekly basis throughout the experiment to avoid confounding location or light effects. Trees were kept this way from March 7th to July 30th. In the Morning (ca. 7:00 am) of the two treatment days, 60 mL of either deionized water, 20 mM 15KNO3 solution or 20 mM 15NH4Cl solution were added to each treatment tree of the control group and the two treatment groups, respectively (120 mL total). Trees were harvested on day 3. RNA was extracted using the CTAB method of Chang et al. (1993). mRNA libraries were constructed using the NEBNext RNA Ultra II Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's protocol.