E. coli BW25113 grown on minimal medium with different carbon sources
E. coli BW25113 grown on minimal medium with different carbon sources
Cells were collected in the exponential growth phase, while at a concentration of ~1.5x10^8 cells/mL. The collection was performed by centrifugation in a microcentrifuge at 20 000 rpm, for 1 min. The supernatant was discarded and the cell pellets flash-frozen in liquid N2. E.coli cells were inoculated in M9 + 2.2 g/L glycerol; M9 + 3.3 g/L sodium pyruvate or M9 + 2.2 g/L glycerol + 2 g/L casamino acids. After the first overnight growth (to saturation), cells were diluted two additional times into pre-heated medium with the same composition: between the 1st and 2nd dilution, the incubation period was ~8hours; between the 2nd dilution and sample collection, the incubation period was ~15h.  For reverse transcription, 2 µg of total RNA were mixed with 1 µL 20 µM random 9N primers, 1 µL TGIRT-III RT (InGex), 4 µL 5X RT Buffer (250 mM Tris pH 8.3, 375 mM KCl, 15 mM MgCl2), 1 µL dithiothreitol 0.1 M and1 µL dNTPs (10 mM each), in a total reaction volume of 20 µL and incubated at 25 °C for 10 min, 57 °C for 1 h. To convert the RNA-DNA hybrid to dsDNA, the TGIRT-III enzyme was first degraded by addition of 1 µg Proteinase K, incubating at 37 °C for 20 min. Then Proteinase K was inactivated by addition of 0.5 µL of Sigma Aldrich Protease Inhibitor Cocktail. The reaction was used as input for the NEBNext Second Strand Module, and second strand synthesis was performed at 16 °C for 1 h, as per manufacturer instructions. dsDNA was tagmented using the Illumina Nextera XT kit, as per manufacturer instructions.