NextSeq 500 sequencing; Comparative transcriptome analysis of a panel of Escherichia coli natural isolates
Comparative transcriptome analysis of a panel of Escherichia coli natural isolates
Overnight cultures of the 16 strains were diluted 100-fold in 1 mL of LB in a 96-deepwell plate (Masterblock 96 well, 2ml, V-bottom plates by Greiner Bio-one). After 2.5 h at 37°C, cultures were diluted to OD ~ 0.02 in 1.4 mL of LB and were further grown for 2 h at 37°C on a Thermomixer (Eppendorf) with shaking (700 rpm). Each culture was then transferred to a 2-mL Eppendorf containing 170 µL of stop solution (5% acid phenol in ethanol) and cooled down for 10 seconds in a bath of dry ice and ethanol. Cells were harvested by centrifugation at 0°C (1 min – 16,000 g) and the pellets were frozen at -80°C.  For RNA extraction, pellets were thawed on ice, resuspended in 200 µL of pre-warmed lysozyme solution and incubated for 3 min at 37°C before addition of 1 mL of Trizol. Samples were vigorously vortexed and incubated at room temperature for 5 min followed by addition of 200 µL of chloroform. After vigorous vortexing, samples were incubated for 5 min at room temperature and centrifuged (10 min – 12,000 g) to separate phases. The upper aqueous phase was collected and RNA was precipitated by addition of 500 µL of isopropanol. Samples were incubated for 10 min at room temperature before centrifugation (10 min – 12,000 g). Pellets were washed with 1 mL of 75% ethanol and centrifuged (5 min – 7,500 g). The pellets were finally air dried, and resuspended in 50 µL of pure water. RNA samples were DNAse-treated using TURBO DNA-free Kit (Thermo Fisher Scientific) and sample quality was assessed on an Agilent Bioanalyzer 2100. Samples were prepared for sequencing using the TruSeq® Stranded Total RNA Kit (Illumina).