RNA-seq of E.coli under regulated and disrupted growth arrests - first repetition
RNA-seq of E.coli under regulated and disrupted growth arrests - first repetition
Each biological replicate is inoculated from a frozen aliquot of a different biological replicate as described in \"biological replicates\". Cultures are grown to regulated or disrupted growth arrest as described in \"growth and growth arrest conditions\". Samples of growth phase were collected just before SHX adition. Cultures were kept in growth arrest for 24h before sample collection. Samples of 1.8ml were collected from each biological replicate. Technical replicates are different samples collected from a single biological replicate (a single culture). The samples are taken to 5 minutes of spin down in 5000g in 4°. After spin down the excess fluid is discarded, resuspended in 50μl TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA), moved into a precooled Eppendorf with 5μl of 9mg/ml Lysozyme in RNase-free water (Sigma), quickly thrown into liquid nitrogen, and stored at −80°C.  The frozen samples were subjected to two cycles of thawing at 37°C and refreezing in liquid nitrogen. Next, the samples were resuspended thoroughly to homogenization with 1 mL TriReagent (prewarmed to room temperature) and incubated for 10 min at room temperature. Two hundred microliters of chloroform were added, and the tubes content was mixed by inversion for 15 s. The samples were incubated for 10 min at room temperature, centrifuged (14,000rcf, 10 min at 4°C) and the upper phase was collected and transferred into new Eppendorf tubes. For RNA precipitation, 500 μl isopropanol was added, the tube contents were mixed thoroughly by inversion and incubated for 10 min at room temperature. The tubes were centrifuged (14,000rcf,15 min at 4°C) and the supernatant was discarded. The pellets were washed twice by addition of 1 mL of freshly made 75% (vol/vol) ethanol (made with DEPC treated water and kept on ice), followed by centrifugation (14,000rcf for 5 min at 4°C) and removal of the supernatant. Next, samples were spin down for 3 min at room temperature, and the access fluid was discarded. Pellets were dried by leaving the tubes open for 10-15 min at room temperature, and then re-suspended in 50 μL RNase-free water and stored at −80°C. RNA quality and concentration was assessed by Nanodrop (ThermoFisher Scientific) and by Bioanalyzer (Agilent).  rRNA depletion, library preparation and RNA-sequencing was done by the center for genomic technologies, the Institute of life sciences, the Hebrew University of Jerusalem. rRNA depletion was done using NEBNext rRNA depletion kit for bacteria. Library preparation was done using NEBNext ultra II directional RNA library prep kit.