title: GSM7964811: early stationary NaCl 5; Clostridioides difficile; RNA-Seq
library_construction_protocol: At the appropriate time points, 5mL aliquots of the cultures were diluted 1:1 in chilled 1:1 Ethanol:Acetone to preserve RNA [45]. These samples were then centrifuged for 5 minutes at 3000 x g at room temperature and cell pellets were frozen at -80˚C. Immediately prior to RNA extraction, cell pellets were centrifuged at 4˚C at 3000 x g for 1minute. Residual supernatant was removed from the cell pellets, which were subsequently washed with 5mL nuclease free PBS. Washed pellets were centrifuged at 4˚C at 3000 x g for 1minute, the supernatant was removed, and the resulting pellet was resuspended in 1mL TRIzol and processed using a TRIzol Plus RNA Purification Kit (Thermo) with on-column DNase treatment according to the manufacturer's instructions. Purified RNA was frozen at -80C and RNA integrity was confirmed via BioAnalyzer (Agilent) prior to proceeding. Library preparation was performed using Illumina's Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit and 10bp IDT for Illumina indices.