title: Illumina HiSeq 2000 sequencing; RNA-seq transcriptome profiling in the Fagus sylvatica trunk
design: RNA-seq transcriptome profiling in the Fagus sylvatica trunk
library_construction_protocol: A 100 cm^2 area of bark was peeled of a living tree. Developing xylem was scraped from the surface underneath and directly frozen in liquid nitrogen. Mint-2 Kit for cDNA preparation (Evrogen) & cDNA normalization TRIMMER kit (Evrogen) Frozen tissue was ground under liquid nitrogen in a ball mill (Retsch, Haan, Germany). Tissue powder (500 mg) was used for RNA extraction after Chang et al. (1993) with minor modifications: the extraction buffer contained 2% b-mercaptoethanol and no spermindine. RNA was purified (RNeasy Mini Kit, Qiagen, Venlo, Netherlands). Total RNA yield and purity were determined spectrophotometrically (NanoDrop, Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). TruSeq RNA Library Preparation Kit (Illumina)