GSM2176845: RNase_E-TS_28C_R1; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344; RNA-Seq
Bacterial cultures were mixed with 0.2 vol. STOP solution (95 % Ethanol, 5 % Phenol) and subjected to RNA extraction following the hot phenol protocol. In brief, bacteria were lysed at 64°C in 600 µl of lysozyme solution (0.5 mg/ml, pH 8.0) and 60 µl of 10% SDS. After adding 66 µl of 3 M NaOAc (pH 5.2), the lysate was extracted by phenol and followed by a chloroform extraction. Total RNA was precipitated in ethanol at -20°C, and subsequently treated with 5 units of DNase I at 37°C for 45 min. For in vitro assays, RNA were extracted using phenol:chloroform:isoamylalcohol (P:C:I, 25:24:1) and precipitated in ethanol at -20°C. Total RNAs were converted to cDNA libraries at vertis AG (Munich, Germany) following a standard protocol described previously (Chao et al. EMBO J, 2012; Westermann et al. Nature, 2016). In brief, RNA was poly(A)-tailed using poly(A) polymerase, and the 5’PPP structure was removed using tobacco acid pyrophosphatase (TAP, epicenter). An RNA adaptor was ligated to the 5’P of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase, and purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics).
GEO Accession
GSM2176845