Large Scale RNA Extraction
200 ml of E. coli culture was pelleted at 10000 xg for 10
minutes, before extraction by a protocol based upon RNASwift (Nwokeoji
et al., 2016). Cells were suspended in 5 ml of 5 mg/ml lysozyme solution
(Thermo Fisher), and allowed to lyse for 10 minutes at room temperature.
12ml of lysis solution (4% SDS, 0.5 M NaCl (Thermo Fisher)) was added,
before incubation for 5 minutes at 65 ºC. 6.8ml of 5M NaCl of was added,
and suspensions were placed on ice for 5 minutes to promote
precipitation of SDS. Suspensions were centrifuged at 10000 xg for 20
minutes at 4 ºC, and supernatant was transferred to a separate tube. RNA
was precipitated from the supernatant by isopropanol or ethanol, and
stored at -20 ºC.