GSM8645356: Rned500_rep1; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
To extract RNA from plate culture, we collected ~ 3 to 4 ml of soft-agar into 50 ml falcon tube. Bacterial enzymatic activity was stop by incubating in 8 ml of stop solution for 3 min. Soft-agar that contained bacterial cells were pelleted by centrifugation at 12,000 rpm, 5 min, 4 ℃. After that, agar pellets were resuspended in 4 ml of KJ medium and 4 ml of lysis buffer. To lyse the bacteria in soft-agar, the mixture was boiled in hot water for 3 min. Total RNE were extracted by adding 4 ml of acid phenol (pH 4.5) and mixed thoroughly by slowly inverting the tube ~ 30 times. Total RNA were extracted in aqueous phase by centrifugation at 4000 g, 1 h, 4 ℃. Total RNA were precipitated in 1 volume of isopropanol and 1/10 volume of 3 M sodium acetate (pH 7.8) at – 20 ℃ for at least 2 h as described above. All RNA samples were maintained in isopropanol at – 20 ℃ before further experiments. five ug total RNA with ERCC control RNA were prepared for libraries using  Ribominus bacteria 2.0  Kit (ThermoFisher A47335) and Illumina TruSeq total  RNA standard Sample Prep protocol.RNAseq on Illumina NextSeq 500 platform as single read 75 bp.