GSM7950696: Rned500/pFL, bio-repeat 1; Escherichia coli K-12; 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 ℃