Instrument: Illumina MiSeq
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: 6 ml samples were taken from exponential and late stationary phase cultures grown on Glc or SA in MM. In each case, two independent biological replicas were used. The samples were harvested (17,226 rcf, 5 min, 4 °C) and immediately frozen in liquid nitrogen and stored at -80°C until RNA isolation. The frozen samples were resuspended in 350 µl Zymo DNA/RNA Shield. The resuspended cells were mixed with 700 µl RLT buffer (Qiagen) (with β-merkaptoetanol) and were transferred into a new tube containing 0.8 g, 0.5 mm Glass Beads (Scientific Industries, SI-BG05). The cells were vortexed for 5 minutes on maximum speed with a Vortex-Genie 2 (Scientific Industries, SI-0236) supplemented with a TurboMix Attachment (Scientific Industries, SI-0564). Following lysis, the mixtures were centrifuged at 9,600 rcf for 10 seconds to remove the glass beads and cell debris. The supernatants were used for purification of total RNAs with the Qiagen RNeasy Mini Kit protocol (Qiagen) with on-column DNase digestion. Then the Purified RNAs were treated again with RNase free DNase I (Sigma) to remove any residual genomic DNA contamination. Finally, the RNAs obtained were repurified with the RNeasy Mini Kit Clean Up protocol (Qiagen). The integrity of the RNA was checked by Agilent 2100 Bioanalyzer. RNA libraries were prepared for sequencing using standard Illumina epicenter ScriptSeq (Bacteria) protocol