RNA-seq analysis of CESA8pro:LUC under different time-points
RNA-seq analysis of CESA8pro:LUC under different time-points
At the onset of peduncle elongation, plants were transferred from long-days conditions with photocycles and thermocycles to the imaging chamber under constant light with thermocycles. The CESA8 bright zone of the peduncle was harvested at seven time points over the course of one day, with four biological replicates per time point. Each replicate comprised three segments collected from the tallest stem of three individual plants. Sampling began near the end of the warm daytime period, 30 min prior to the transition to nighttime temperature (ZT15.5), followed by three time points during the cooler night (ZT16.5, ZT20, and ZT23.5), and three time points after the onset of the warm daytime period, which began at ZT24 (ZT24.5, ZT27, ZT32).  Seeds were imbibed for 21 days at 4°C and then planted in Pro-Mix BX Mycorrhizae potting mix. The seeds were planted 1.5 cm deep in 22 cm tall Cone-tainer pots with a 3.8 cm diameter (Ray Leach SC10R, Stuewe & Sons, Inc.). Plants were first grown in a PGC-105 growth chamber (Percival Scientific, Perry, IA) with a ~450 μmol·m−2·s−1 day period of 16 h at 25°C and a dark night period of 8 h at 17°C and watered daily.  Total RNA was extracted from each time point using the Plant RNeasy mini kit (Qiagen) according to manufacturer instructions, including on-column treatment digestions with RNase-free DNase I.  RNA quality assessment, library preparation, and sequencing were performed by Plasmidsaurus using an Illumina platform with a 3' end counting approach.