Illumina HiSeq 2000 sequencing; Differential RNA-seq for identifying toxin MazF and MqsR cleavage sites, beginning of transcripts and stationary phase cleavage sites in Escherichia coli (mRNA, rRNA and other ncRNA)
Differential RNA-seq for identifying toxin MazF and MqsR cleavage sites, beginning of transcripts and stationary phase cleavage sites in Escherichia coli (mRNA, rRNA and other ncRNA)
Lysogeny broth (LB) and LB agar plates were used for growth. Liquid cultures were grown on Infors HT Multitron shaker, at 190 RPM, at 37C.Overnight cultures were started from a fresh colony and grown for 16-17h. For growing the logarithmic phase and stationary phase samples, the overnight cultures were diluted 1000-fold into 100 ml of LB in 1 l flasks. At OD600nm  0.2, 25 ml of the culture was used for the log phase RNA extraction. The remaining culture was grown for 24 h and used for the stationary phase RNA extraction. The toxin expression was induced in strain BW25113 harboring either plasmids pSC228 and pSC3326 for the induction of MazF and MazE, or pTX3 and pAT3 for the induction of MqsR and MqsA. Ampicillin 30 g/ml or 100 g/ml and chloramphenicol 50 g/ml were used to maintain the plasmids in the culture. The medium of overnight cultures was supplemented with 0.2% glucose to avoid toxin leakage and 1 mM IPTG to induce antitoxin. The overnight cultures were diluted 1000-fold into 200 ml of LB in 1 l flasks. At OD600nm  0.2, 1mM L-arabinose was added to induce the toxin and incubation was continued for 2h.  25-50 ml of culture was used for separating total RNA from log phase and toxin overexpression cultures. Cells were rapidly collected by centrifugation at 20C for 5 min at 5000 x g and frozen in liquid nitrogen. Frozen cells were stored at -80C. 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 67C, and were re-inverted after each minute during the incubation. Lysates were centrifuged for 10 min at 4C 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 4C 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 -20C. 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 - 80C.  For the stationary phase RNA isolation, bacteria were collected from 5 ml samples, washed with 10 ml of 1xPBS before the hot phenol extraction, and MaXtract Low (Qiagen) was used for better phase separation after the phenol:chloroform (1:1) extraction. To construct the 5'-end cDNA library, the total RNA samples were first treated with either tobacco acid pyrophosphatase (Epicentre), T4 polynucleotide kinase (NEB), or left untreated. Afterwards, an RNA adapter was ligated to the 5'-monophosphates of the RNA fragments. First-strand cDNA synthesis was primed with an N6 randomized primer and performed by M-MLV reverse transcriptase. Then, Illumina adapters containing barcode sequences were ligated to the 5'-ends of the antisense cDNA. The adapters had two random nucleotides at the 3-end to diminish the ligation bias. The cDNA was finally amplified with PCR (14 to 18 cycles) using a 5'-biotinylated primer and a proof reading enzyme. For Illumina sequencing, 300  500 bp long 5' fragments were isolated from the full-length cDNAs. For this purpose, the cDNA preparations were degraded using NEBNext dsDNA Fragmentase and the 5'-cDNA fragments were then bound to streptavidin magnetic beads. The bound cDNAs were blunted and the 3' Illumina sequencing adapter was ligated to the 3'-ends of the cDNA fragments. The bead-bound cDNAs were finally PCR-amplified (4-7 cycles).