title: RNA-seq of drought response and drought recovery in barley (cv. Golden Promise) on a lysimeter platform.
design: RNA-seq of drought response and drought recovery in barley (cv. Golden Promise) on a lysimeter platform.
library_construction_protocol: Second leaves from the youngest tillers were collected for RNA analysis: Timepoint 1 (T1) on the last day of the well-watered phase (day 12); T2, on the last day of Dry Phase II (day 41); T3, last day of Rewatering II (day 44). Well-watered control samples at the same growth stages were collected at T2 and T3. For RNA extraction, leaf samples were collected in liquid N2 and later ground and stored in TRIzol reagent (Invitrogen). Total RNA was extracted from the leaf samples with the Direct-zol RNA Miniprep Plus kit according to the manufacturer's (Zymo Research Europe GmbH) guidelines. RNA (1 µg) from each sample was treated with Ambion® DNase I (RNase-free; Applied Biosystems) in a 20 µl reaction mixture for 90 min at 37 °C to remove remaining DNA. An RNeasy MinElute Cleanup Kit (Qiagen) was used to purify and concentrate the RNA from the DNase treatments. RNA quantity and quality were checked both spectrophotometrically (NANODROP-2000 spectrophotometer, Thermo Scientific) and by agarose gel electrophoresis. Plants grown during the winter season in the iCORE functional-phenotyping greenhouse (https://plantscience.agri.huji.ac.il/icore-center) under natural light, with moderate temperature control, in Rehovot, Israel. Seeds were sown in germination trays, then transplanted at the 3-4-leaf stage (two weeks old) to pots. Before transplanting, seedling roots were carefully to remove the original soil, planted into potting soil (“Bental 11”, Tuff Marom Golan, Israel), four per 3.9-litre pot, then placed onto lysimeters. The plants were grown at 18–25°C, 20–35% relative humidity, and 10h light /14h dark. The vapor pressure deficit (VPD) ranged between 0.2 to 4.0 kPa in the greenhouse and represented typical weather fluctuations during winters in Central Israel Drought experiments were carried out on a gravimetric functional phenotyping platform (Dalal A et al. 2020. A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant-Environment Interactions. J Vis Exp. DOI: 10.3791/61280). The experiment was divided into five phases on the lysimeter platform: (i) well-watered; (ii) Dry Phase I (iii); Rewatering I (iv); Dry Phase II; (v) Rewatering II. For the well-watered phase, all plants received daily night irrigation for the first 12 days with three irrigations in 3-hour intervals. In Dry Phase I, the system was set to irrigate each plant to 80% of its own previous day's transpiration, so that all plants would be subjected to the same gradual water stress from day 13 to day 24, followed by rewatering (Rewatering I) on the night of day 25. Dry Phase II extended from day 26 to day 41 with the same irrigation parameters as Dry Phase I, until the time when the droughted plants reached <100g daily transpiration (around 20 to 30% of controls). Rewatering II began on day 42. Daily night irrigation was given to all plants until day 44. On the first irrigation, several irrigation cycles were used to moisten the soil and to ensure a laterally uniform soil water content (SWC) in the pots. VPD data were taken daily at 17:22 from the SPAC analysis. Total RNA was extracted from the leaf samples with the Direct-zol RNA Miniprep Plus kit according to the manufacturer's (Zymo Research Europe GmbH) guidelines. RNA (1 µg) from each sample was treated with Ambion® DNase I (RNase-free; Applied Biosystems) in a 20 µl reaction mixture for 90 min at 37 °C to remove remaining DNA. An RNeasy MinElute Cleanup Kit (Qiagen) was used to purify and concentrate the RNA from the DNase treatments. RNA quantity and quality were checked both spectrophotometrically (NANODROP-2000 spectrophotometer, Thermo Scientific) and by agarose gel electrophoresis. Libraries were constructed using the TruSeq Stranded Total RNA kit; adaptors were TruSeq adaptors. Cytoplasmic and chloroplast was removed by hybridization on beads. Following rRNA removal, the RNA was fragmented into small pieces using divalent cations under elevated  temperature. Cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers.