Illumina HiSeq 2000 paired end sequencing; Senescence time course of unfertilized Arabidopsis thaliana ovules
Senescence time course of unfertilized Arabidopsis thaliana ovules
After around 35 days of growth all stage 12c flowers on the main stem were emasculated and harvested after 2, 3, or 4 days.  Unpollinated pistils of emasculated wild-type flowers were dissected as described previously. For each time point the ovules of over 75 pistils (approximately 4000 ovules) were collected on dry ice with the help of a vacuum-driven micro-aspirator (Sanchez-Leon et al., 2012). The whole time series was sampled in three independent batches (four time points in each round, 2 to 4 DAE).   Arabidopsis seeds (Columbia-0, Col-0) were sown on Jiffy-7C propagation plugs (Jiffy) and stratified for two to three days at 4 °C. Plants were grown for about 35 days at 22°C under a 16 h light/8 h dark regime before being emasculated. After subsequent tissue grinding, RNA was extracted with the Spectrum Plant Total RNA kit (Sigma). RNA quantity and quality was assessed by spectrophotometric analysis (NanoDrop ND-2000, Thermo) and on-chip electrophoresis (RNA 6000 Nano Kit, Agilent).  Library preparation was done using the TruSeq unstranded RNA Sample Preparation Kit v2 (Illumina).