characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-02-01T00:19:09Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-02-01T00:19:09Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112239908
characteristics.characteristic: External Id
characteristics.text: The Hebrew University of Jerusalem
characteristics.characteristic: INSDC center name
characteristics.text: 2023-02-01T00:19:09Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-02-01T00:19:09Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14350795
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12466:Sample 12
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: Protocols: As a model sRNA, we use the well-known sRNA GcvB, a sRNA that regulates genes involved in amino acid metabolism and transport. To study the cooperation between GcvB and RNase E we apply the mutant cycle approach to four strains of E. coli: wt; GcvB mutant/RNase E wt; GcvB wt/RNase E mutant; GcvB mutant/RNase E mutant. Bacteria were centrifuged for 5 min at 4°C,4500g, resuspended in 50 µl TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) and mixed with 5 ul of 9 mg/ml lysozyme prior to freezing the sample at liquid nitrogen and storing it at -80°C. Single colonies were grown in LB-Amp at 30°C while shaking over night. The next day, each culture was diluted 1:100 into two flasks containing fresh LB-Amp and grown at 30°C to an OD600 of 0.3. Then, one flask was transferred to 44°C while the other one was left at 30°C. After 20 min both cultures were treated with IPTG at a final concentration of 1 mM, and left for an additional 10 min at the respective temperatures. Cultures were collected. Frozen samples were subjected to two cycles of thawing at 37°C and refreezing in liquid nitrogen. Next, the samples were re-suspended thoroughly to homogenization with 1 mL TriReagent (pre-warmed to room temperature) and incubated for 5 min at room temperature. Two hundred microliters of chloroform were added, and the tubes content was mixed by inversing the tubes for 15 s. The samples were incubated for 10 min at room temperature, centrifuged (17,000g, 10 min at 4C) and the upper phase was collected and transferred into new Eppendorf tubes. For RNA precipitation, 500 l isopropanol were added, the tube contents were mixed thoroughly by inversion of the tubes and incubated for 10 min at room temperature. The tubes were centrifuged (17,000g,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, followed by centrifugation (17,000g, 5 min at 4°C) and removal of the supernatant. Pellets were air dried by at room temperature, and then re-suspended in 300 L nuclease free water and stored at -20°C. The RNA concentration was measured using Nanodrop (ThermoFisher Scientific). tagRNA-seq analysis of the transcriptional organization of a bacterium by vertis Biotechnology AG- tagRNA-seq is a modified RNA-seq method which is based on the differential labelling of 5' RNA ends. It enables to discriminate primary from processed 5' RNA ends. The method is described in detail by Innocenti, N. et al. 2015. RNA (New York, N.Y.). 21, 5 (May 2015), 1018-30 It is based on specific ligation of distinct sequence tags to processed start sites (PSS) and transcription start sites (TSS), thereby gaining insight into regulatory processes at transcriptional and post-transcriptional levels.
characteristics.characteristic: description
characteristics.text: RNAseE 3071 loss of function mutation
characteristics.characteristic: genotype
characteristics.text: LB-Amp broth
characteristics.characteristic: growth condition
characteristics.text: Escherichia coli str. K-12 substr. MG1655
characteristics.characteristic: organism
characteristics.text: Escherichia coli str. K-12 substr. MG1655
characteristics.characteristic: organism
characteristics.text: whole organism
characteristics.characteristic: organism part
characteristics.text: RNA-seq
characteristics.characteristic: protocol
characteristics.text: Escherichia coli str. K-12 substr. MG1655
characteristics.characteristic: scientific_name
characteristics.text: MG1655
characteristics.characteristic: strain
characteristics.text: 44
characteristics.unit: Celsius
characteristics.characteristic: temperature
characteristics.text: Sample 12
characteristics.characteristic: title