title: RNA-seq files of Virulent and Avirulent EPEC microcolonies from aerobic, butyrate, infant stool metabolites and anaerobic conditions
library_construction_protocol: EPEC wild type E2348/69 (strain O127:H6) bacteria were grown overnight (ON) in LB medium (Sigma-Aldrich) at 37°C without shaking, diluted 1:40 into DMEM-HEPES (Gibco, Israel) medium and incubated for 3h at 37°C without shaking to exponential phase (optical density (OD) ~ 0.6). Bacteria were plated on LB plate with streptomycin (50 µg/ml), incubated at 32°C for 15h (for aerobic LB) or for 12h (for aerobic butyrate and infant stool metabolites conditions) or at 37°C (for anaerobic condition) for 12 h and colonies of two sizes (Virulent & Avirulent) were sampled using a sterile picking stick and resuspended in 100 μl ice-cold TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA). Ten percent of each microcolony was resuspended in glycerol (15% final concentration) and stored at −80°C for further analysis. Lysosyme (0.9 mg/ml, Sigma-Aldrich) was added to the remaining sample and the samples were frozen at liquid nitrogen and stored at −80°C. The frozen samples were subjected to two cycles of thawing at 37°C and refreezing in liquid nitrogen. Next, the samples were resuspended thoroughly to homogenization with 1 mL TriReagent (prewarmed to room temperature) and incubated for 5 min at room temperature. Two hundred microliters of chloroform were added and the tubes content was mixed by inversing the tubes for 15 s. The samples were incubated for 10 min at room temperature, centrifuged (17,000g, 10 min at 4°C) and the upper phase was collected and transferred into new Eppendorf tubes. For RNA precipitation, 500 μl isopropanol was added, the tube contents were mixed thoroughly by inversion of the tubes and incubated for 10 min at room temperature. The tubes were centrifuged (17,000g,15 min at 4°C) and the supernatant was discarded. The pellets were washed twice by addition of 1 mL of freshly made 75% (vol/vol) ethanol, followed by centrifugation (17,000g for 5 min at 4°C) and removal of the supernatant. Pellets were dried by leaving the tubes open for 15 min at room temperature, and then re-suspended in 300 μL nuclease free water and stored at −20°C. The RNA concentration was measured using Nanodrop (ThermoFisher Scientific). rRNA depletion was done by using the DIY rRNA depletion method (Culviner et al., 2020), using the rRNA sequence probe set designed for E. coli K-12. RNA-seq libraries were constructed based on the RNAtag-Seq protocol (Shishkin et al., 2015) with several modifications (Melamed et al., 2018). Since the rRNA depletion step was done in advance, after the fragmentation step the first ligation was carried out.