RNA-seq protocol for transcriptomic analysis

RNA-seq was performed using two biological replicates (Dataset S5). The strains were grown under the same conditions as those used in the ChIP-exo experiments. Transcripts were stabilized by mixing 3 ml of cell cultures at the mid-log phase with 6 ml of RNAprotect Bacteria Reagent (Qiagen). Samples were immediately vortexed for 5 s, incubated for 5 min at room temperature, and then centrifuged at 5000 × g for 10 min. The supernatant was decanted, and any residual supernatant was removed by inverting the tube once onto a paper towel. Total RNA samples were then isolated using a RNeasy Plus Mini kit (Qiagen) following the manufacturer's instruction. Samples were then quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific) and quality of the isolated RNA was checked by running RNA 6000 Pico Kit using an Agilent 2100 Bioanalyzer (Agilent). Paired-end, strand-specific RNA-seq libraries were prepared using KAPA RNA Hyper Prep kit (KAPA Biosystems), following the instructions (20 (link),21 (link)). Resulting libraries were analyzed on an Agilent Bioanalyzer DNA 1000 chip (Agilent). Sequencing was performed on a Hiseq 2500 sequencer (illumina) at the Genomics Core facility of University of California, San Diego.