Measuring the effects of pervasive transcription on promoter activity in Salmonella Pathogenicity Island 1 by semiquantitative 5'RACE-Seq
Measuring the effects of pervasive transcription on promoter activity in Salmonella Pathogenicity Island 1 by semiquantitative 5'RACE-Seq
Overnight bacterial cultures in LB broth were diluted 1:200 in the same medium - or in LB broth supplemented with 0.1% ARA or 0.4 µg/ml Anhydrotetracycline (AHTc) or both drugs where appropriate - and grown with shaking at 37°C to an OD600 = 0.7 to 0.8. Cultures (4 ml) were rapidly centrifuged and resuspended in 0.6 ml ice-cold REB buffer (20 mM Sodium Acetate pH 5.0, 10% sucrose).  RNA was purified by sequential extraction with one volume (0.6 ml) of hot acid phenol, phenol-chloroform 1:1 mixture and chloroform. Following overnight ethanol precipitation at -20°C and centrifugation, the RNA pellet was resuspended in 20 µl of H2O. Three samples were prepared from independent biological replicates for each strain and condition. RNA yields, measured by Nanodrop reading, typically ranged between 2 and 3 µg/µl. RNA (1 µg) was combined with 1 µl of a mixture of up to four gene-specific primers, 5 µM each (including AI41 (hilD), AI48 (prgH) and AJ33 (ompA)), and 1 µl of 10 mM dNTPs in a 6 µl final volume. After a 5 min treatment at 70°C (in a Thermocycler), samples were quickly cooled on ice. Each sample were then mixed with 2.5 µl of Template Switching Buffer (4x), 0.5 µl of 75 µl of Template Switching Oligonucleotide (TSO) and 1 µl of Template Switching RT Enzyme Mix in a final volume of 10 µl. Reverse transcription was carried out for 90 min at 42°C, followed by a 5 min incubation at 85°C. The cDNAs were amplified in parallel with a common forward primer (AJ38, annealing to the TSO) and a reverse primer specific for the region being analyzed and carrying a treatment-specific index sequence. PCR reactions were set up according to New England Biolabs PCR protocol for Q5 Hot Start High-Fidelity DNA polymerase in a final volume of 50 µl (using 1 µl of the above cDNA preparation per reaction).