control
Escherichia coli str. K-12 substr. MG1655
Protocols: 20-50 ml of culture was used for separating total RNA from log phase and toxin overexpression cultures. Cells were rapidly collected by centrifugation at 20°C for 5 min at 5000 x g and frozen in liquid nitrogen. Frozen cells were stored at -80°C. Bacteria were grown in lysogeny broth (LB) and on LB agar plates. Liquid cultures were grown at 37°C with aeration at 190 RPM on Infors HT Multitron shaker. Ampicillin (30 μgml-1 or 100 μgml-1) and chloramphenicol (50 μgml-1) were added for plasmid maintenance as required. Escherichia coli MG1655 was used as the control for RNA sequencing. Overnight culture was started from a fresh colony, grown for 16 to 17h, and diluted 1:1000 into 100 ml in 1 l flask. At OD600 ≈ 0.2, 50 ml of the culture was used for RNA extraction. The toxins were induced in BW25113 harboring either plasmids pSC228 for MazE expression and pSC3326 for MazF expression or pAT3 for MqsA expression and pTX3 for MqsR expression. The overnight culture medium was supplemented with 0.2% glucose to avoid the toxin leakage and 1 mM IPTG to induce the antitoxin. The overnight culture was diluted 1:1000 into 200 ml of LB in 1 l flasks. At OD600nm ≈ 0.2, 50 ml of the culture was removed and L-arabinose was added to final concentration of 1 mM to induce the toxin expression. After 2h, 20 ml of the culture was used for RNA extraction. At the same time, 50 ml of culture was collected by centrifugation (20°C for 5 min at 5000g), re-suspended in 1 ml of LB and added to 50 ml pre-warmed medium in 1 l flask. After 30 min recovery, 20 ml of the culture was used for RNA extraction. For RNA isolation cells were resuspended in 600 ul of ice-cold 1x TEN solution (10 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl) and added quickly to the 600 ul preheated phenol-1% SDS solution (pH 4.5). Samples were mixed gently by inverting tubes a few times, incubated for 5 min at 67°C, and were re-inverted after each minute during the incubation. Lysates were centrifuged for 10 min at 4°C at 15 700 × g. Since here, all preparations were done on ice. 450 ul of aqueous phase was collected and extracted with 600 ul of phenol:chloroform, mixed vigorously on vortex, centrifuged as above and further extracted with 500 ul of chloroform. After centrifugation for 1 min at 4°C at 15 700 × g, 350 ul of aqueous phase was allocated, 0.1 volumes of 3M RNase free sodium acetate (pH 5.2) was added, and RNA was precipitated with 1.2 volumes of ice-cold isopropanol during 30 min incubation at -20°C. Tubes were then centrifuged for 40 min at 15 700 × g, RNA pellets were washed with 700 ul of 75% ethanol and centrifuged for 5 min at 12 000 × g. Pellets were air dried at room temperature for 10 min, dissolved in ddH20 and stored at - 80°C. Total RNA was treated with DNase and rRNA was depleted using the Ribo-Zero rRNA Removal Kit (Bacteria, Epicentre). RNA was fragmented with ultrasound (1 pulse of 15 sec at 4°C), followed by first-strand cDNA synthesis primed with N6 randomized primers. Illumina TruSeq sequencing adapters were ligated to the 5' and 3' ends of the cDNA. The cDNA was amplified with PCR (18 cycles). For Illumina sequencing, the samples were pooled in approximately equimolar amounts and the DNA fragments in the size range of 500 - 800 bp were eluted from a preparative agarose gel. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina.
ENA-FIRST-PUBLIC
2018-09-18T09:52:43Z
ENA-LAST-UPDATE
2018-07-09T11:32:19Z
External Id
SAMEA4780012
INSDC center name
Institute of Technology, University of Tartu
INSDC first public
2018-09-18T09:52:43Z
INSDC last update
2018-07-09T11:32:19Z
INSDC status
public
Submitter Id
E-MTAB-6993:control
broker name
ArrayExpress
genotype
wild type genotype
growth condition
logarithmic phase culture
sample name
E-MTAB-6993:control
scientific_name
Escherichia coli K-12
strain
K-12
substrain
MG1655