GSM9014281: ΔUhpTU + pBAD-UhpU-EC-G6P R2; Escherichia coli; RNA-Seq
Bacterial total RNA was isolated using the hot phenol method. Briefly, Cells were resuspended in lysozyme, 10% SDS and 3M sodium acetate (pH5.2). The cleared lysate was mixed with saturated phenol (pH4.5) and incubated at 64°C for 6 min with shaking. After mixing with chloroform and centrifugation in a Phase Lock Gel tube (#WM5-2302820, TIANGEN Biotech), the aqueous phase was collected and mixed the with 30:1 ethanol: sodium acetate (pH 6.5) and precipitated at -80°C overnight. RNA pellets were washed with 80% ethanol and dissolved in ultra-pure water (#10977, Thermo Fisher Scientific), and the RNA concentration was determined 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