Illumina HiSeq 4000 paired end sequencing; RNA-seq of a time series experiment of leaf 3 tissue (primordia / leaf blade) of Arabidopsis plants grown under EoD FR since day 6, since day 18 or under WL conditions.
RNA-seq of a time series experiment of leaf 3 tissue (primordia / leaf blade) of Arabidopsis plants grown under EoD FR since day 6, since day 18 or under WL conditions.
Samples were collected at ZT22-23 into a falcon tube with RNA later solution. Once all samples were collected, L3 primordias and L3 blades were dissected with a scalpel, in a Petri dish filled with RNA later solution, under a Leica MZ 16 F dissecting microscope. Seeds were stratified in a 0.1% Agar solution, in darkness for 5 days at 4°C. Seeds were then sown on F2 + S Levington Advance Seed and Modular Compost plus Sand soil mix (ICL Specialty Fertilizers, Suffolk, U.K.) and grown inside a Percival SE-41L cabinet (CLF Plant Climatics, Wertingen, Germany) under a light : dark (LD) 12 h : 12 h photoperiod, at a 100 µmoles/m2/s fluence rate and 21 °C of constant temperature. On the 6th day, plants were thinned to a density of 1-2 plants per pot. Specific details on growth conditions are mentioned in the respective figure legends. Unless otherwise mentioned, Polylux XLR FT8/18W/835 fluorescent tubes (GE) were used as a white light (WL) light source. For End of Day (EoD) Far-red (FR) treatments, we custom modified the bottom shelf of a Percival cabinet and mounted 7 24V OSLON 150 ILS-OW06-FRED-SD111 FR led strips (Intelligent Led Solutions, Berkshire, UK), to deliver 40µmoles/m2/s of FR light (730nm) for 10 minutes.  Total RNA from was extracted using the RNeasy Plant Mini Kit (Qiagen) with on-column DNase digestion. Samples were then sent to Edinburgh Genomics for QC check and sequencing. Briefly, quality check of the samples was performed using Qubit with the broad range RNA kit (Thermo Fisher Scientific) and Tapestation 4200 with the RNA Screentape for eukaryotic RNA analysis (Agilent). Libraries were prepared using the TruSeq Stranded mRNA kit (Illumina), and then validated.