GSM7502661: △fnr, Red 40, stationary, rep 3; Escherichia coli; RNA-Seq
1 μl of TRI Reagent (Sigma Aldrich catalog number T9424) was added to bacterial pellets and incubated for 10 mins, samples were then transferred to 2 ml Lysing Matrix E tubes (MP Biomecials, catalog number 116914050). Cells were lysed for 5 mins in the bead beater at room temperature. 200 μl of chloroform was added. Samples were vortexed for 15 sec and incubated at room temperature for 10 mins. Samples were then centrifuged at 16,000 x g for 15 mins at 4°C. 500 μl of the upper aqueous phase was transferred to a new tube, 500 μl of 100% ethanol added, and vortexed to mix. Mixture was transferred to a spin column (PureLink RNA Mini Kit; Life Technologies; catalog number 12183025) and centrifuged at ≥12,000 x g for 30 sec, discarding flow-through, until all of the material had been added to the column. To the spin column, 350 µl Wash Buffer I (PureLink RNA Mini Kit; Life Technologies; catalog number 12183025) was added, then the column was centrifuged at ≥12,000 x g for 30 sec, discarding flow-through. 80 μl of PureLink DNase mix was added to the column and incubated at room temperature for 15 mins. 350 μl of Wash Buffer I (Purelink RNA mini kit) was added and the column spun at >12000 x g for 30 sec. The column was transferred to a new collection tube, and 500 µl Wash Buffer II was added, followed by centrifugation at ≥12,000 x g for 30 sec, discarding flow-through. The column was centrifuged at ≥12,000 x g for 60 sec and dried, then moved to a collection tube. 50 µl RNase-free water was added, and the column was incubated at room temperature for 1 min. Finally, the column was centrifuged for 1 min at ≥12,000 x g, retaining the flow-through, which contained total RNA. Samples were DNase treated again using TURBO-DNase (Ambion; ThermoFisher catalog number AM2238) and incubated at 37°C for 30 mins. RNA Ampure XP Beads were used to clean up the reactions. 1.8 volumes of the RNA Ampure XP beads were added to 1 volume of RNA sample and allowed to sit for 5 mins at room temperature. Tubes were placed on a magnetic stand until the liquid cleared. Then liquid was removed and beads washed with 200 μl of 100% Ethanol. Samples were incubated for 30 sec, then removed. This was repeated twice more. Samples were allowed to dry for 5 mins, removed from the magnetic stand, and 30 μl of RNase-free water was added. This was incubated for 2 min at room temperature then placed back on the magnetic rack where liquid was collected after turning clear.