GSM5751885: ΔfadBA ΔFadZ + pBAD-FadZ - Rep1; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344; RNA-Seq
Bacterial cultures were mixed with 0.2 (v/v) of stop solution (95% ethanol and 5% phenol) and immediately frozen within liquid nitrogen. Total RNA was isolated using the hot phenol method. Briefly, 2.0 OD600 cell pellet was resuspended with 300 μL lysozyme (#L3790, Sigma-Aldrich) at 0.5 mg/mL, 30 μL 10% SDS and 33 μL 3M sodium acetate (pH5.2). Cleared lysate was mixed with 375 μL saturated phenol (pH4.5) and incubated at 64°C for 6 min with shaking. The aqueous phase was collected after centrifuge and mixed with 375 μL chloroform in Phase Lock Gel tube (#WM5-2302820, TIANGEN Biotech). RNA was precipitated at -80 °C overnight by mixing the aqueous phase (~350 μL) with 700 μL 30:1 ethanol: sodium acetate (pH 6.5) mix. RNA pellets were washed with 80% ethanol and dissolved in ultra-pure water (#10977, Thermo Fisher Scientific), and was quantified using NanoDrop 2000. Ribosomal RNAs was removed by Biotin-labeled rRNA probe. RNA was fragmented with BGI fragmentation buffer and was reverse transcribed with random hexamer. Double chain cDNA was then blunted and phosphorylated for the 5' end, and overhang A base in the 3' end and ligated with T-overhang adapter. After PCR application, samples were sequenced in BGISeq-500 platform