Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344
Protocols: Overnight cultures were grown from a single colony, diluted 1:100 in fresh LB medium, and grown to the OD600 ~ 0.3 at 37°C with shaking at 220 rpm.Cells were aerobically cultured to an OD 600 of 0.3, filled in 50 ml closed Falcon tubes, and incubated without agitation at 37°C for 30 mins. 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-labled rRNA probe. Purified and fragmentated RNA was then treated by Illumina TruSeq Standard Kit: Fragmented RNA was reverse transcribed to first chain cDNA random oligo and reverse transcriptase. Second chain cDNA was generated with DNA polymerase I and RNase H. dTTP was replaced by dUTP when Second chain cDNA was generated to increase the chain-specificity. Double chain cDNA was then poly A tailed and ligated with adaptor.