GSM5134346: Sample 44 Rehyd 48h PST; Xerophyta schlechteri; RNA-Seq
Total RNA was extracted from hydrated, dehydrating (75%, 55%, 35%), desiccated (5% relative water content (RWC)) and rehydrated (48h – varying RWC values) leaf tissues of X.schlechteri. A modified extraction method was used, using Tri-reagent (Sigma-Aldrich Corporation, St. Louis, MO, USA) and the RNEasy mini-kit (Qiagen, Hilden, Germany). Samples held in storage at-80⁰C were transferred under liquid nitrogen into sterilized 2mL Eppendorff tubes containing three stainless steel bearings (pre-treated by soaking in chloroform and autoclaved twice along with the tubes). Before the tissue was allowed to thaw, 1mL cold Tri-reagent was pipetted into the tubes, ensuring that all tissue was completely submerged. The samples were maintained on ice until being transferred to an in-house homogenizer, where they were homogenized at room temperature for 12 minutes. The samples were placed on ice and 200µl chloroform was added to each tube. The samples were mixed by inversion and maintained on ice for 2 min. The tubes were then centrifuged at 13000g for 15 min at 4⁰C. The upper aqueous phase of each sample was transferred to the RNEasy mini-kit Shredder column and the manufacturer's instructions for RNA extraction were followed with the following modifications: the RW1 buffer wash step was repeated with a volume of 200µl, the RPE buffer wash step was repeated twice and the RNA was eluted in RNase-free water pre-heated to 55⁰C after incubating for 10 min at room temperature. RNA integrity and quality were verified using a Bioanalyzer (Agilent technologies, USA). Library preparation was conducted using the Illumina RNA-seq kit (San Diego, California) according to the manufacturer's instructions.