GSM8218823: SDT387, timepoint2, rep1; Escherichia coli K-12; RNA-Seq
All transcriptome samples were collected and prepared in biological duplicates, whereby either 2 mL of culture (low cell density culture, batch phase) or 0.5 mL of culture (high cell density culture, fed batch phase) was added to 6 mL of Qiagen RNA-protect Bacteria Reagent immediately after sample collection. This solution was then vortexed for 30 seconds, incubated at room temperature for 5 minutes, and then centrifuged. The supernatant was then removed and the cell pellet was stored at -80°C. The RNase-Free DNase Set (Qiagen) was used to extract and purify RNA from the cell pellets per vendor protocol, including on-columns DNase treatment for 30 minutes at room temperature. RNA was quantified using qubit fluorometer (Thermofisher) and integrity was assessed using fragment analyzer (Agilent Technologies). Ribosomal RNA was removed using QIAseq FastSelect –5S/16S/23S kit (Qiagen). Sequencing libraries were prepared with the KAPA Stranded RNA-Seq Library Preparation Kit (Kapa Biosystems) and assessed using fragment analyzer (Agilent Technologies). The libraries were then subjected to paired end sequencing on Illumina NovaSeq.