title: Illumina HiSeq 2000 sequencing; Whole transcriptome analysis in nicotiana benthamiana WT and transgenic lines presenting spontaneous systemic posttranscriptional silencing (PTGS) of a GFP transgene (line GFP6.4 described in Kalantidis Plant J 2006). Change in gene expression was analysed in WT versus GFP6.4 before initiation, during spreading and maintenance of GFP-PTGS.
design: Whole transcriptome analysis in nicotiana benthamiana WT and transgenic lines presenting spontaneous systemic posttranscriptional silencing (PTGS) of a GFP transgene (line GFP6.4 described in Kalantidis Plant J 2006). Change in gene expression was analysed in WT versus GFP6.4 before initiation, during spreading and maintenance of GFP-PTGS.
library_construction_protocol: Leaf tissues were excised and flash frozen in liquid nitrogen plants were grown in controlled environment at 22°C with a photoperiod of 16h light / 8h dark and a photosynthetically active radiation of 90 µmol m-2 sec-1 provided by cool white fluorescent tubes. GFP fluorescence was monitored using a hand-held 1 000 W long-wavelength UV lamp (B1000 AP; Ultraviolet Products, Upland, CA, USA). Total RNA were extracted from leaf tissues grinded in liquid nitrogen using the TRIzol™ (Invitrogen™, ThermoFisher Scientific, Waltham, MA, USA) reagent method following manufacturer instructions. For each RNA samples (WT, GFP6.4, NS, OS and SS), librairies were were constructed using 3µg of pooled total RNAs extracted from four individual plants. Library preparation and sequencing were performed at GenXPro (GmbH, Frankfurt am Main, Germany) as described in (Zawada et al., 2014 Massive analysis of cDNA ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease).
attributes.tag: Experimental Factor: genotype
attributes.value: GFP6.4; Phenotype OS