alias: mycoplasma heatshock
title: Mycoplasma gallisepticum 5`-ERS heatshock
design: Heatshock 46°C for 15 min.  Preparation of 5`-ERS libraries was done as described previously [REF]. Cells from 1 ml of culture were centrifuged for 10 min at 8000 g, resuspended in wash buffer (100 mM Tris, 100 mM NaCl, 2 mM MgCl2, pH 7.4) and lysed in TRIzol LS reagent (Life Technologies) at a 1:3 ratio of resuspended cells:TRIzol LS (v/v). The lysates were extracted with chloroform, and the aqueous phase was purified with a PureLink RNA Mini Kit (Ambion) to remove tRNA or was used directly to precipitate RNA by the addition of an equal volume of isopropanol.  About 20 μg of total RNA was fragmented into 200 bp by chemical fragmentation (100 mM ZnSO4, 100 mM Tris, pH=7.0 at 70°C for 15 min). The fragmentation reaction was stopped with 20 mM EDTA (pH=8.0). The fragmented RNA was end-repaired with T4 polynucleotide kinase according to the manufacturers protocol (Thermo).   Fragmented end-repaired RNA was treated with Terminator exonuclease (Epicentre). This process resulted in the degradation of the non-primary 5’-end RNA fragments, whereas the primary 5’-fragments were protected by the tri-phosphate groups on their 5’-ends. As chemical fragmentation leaves phosphates randomly on fragments ends, end-repair procedure was used to enhance degradation of non-primary 5`-end fragments (e.g. with 5`-OH), which otherwise undergo adapter ligation and cDNA synthesis (if they have 3`-OH) and to rescue primary 5`-end fragments with 3`-phosphate (by 3’-phosphatase activity of T4 PNK). Addition of end-repair procedure increases signal (e.g. coverage of primary 5`-ends) and decreases background. Then, the RNA was precipitated by isopropanol and treated with tobacco acid phosphatase (Epicentre) to remove the pyrophosphate groups. Next, the RNA was precipitated by isopropanol and used for strand-specific ds-cDNA preparation according to the standard protocol for SOLiD libraries. The sample cDNA was normalized in one round as described above and used to prepare SOLiD libraries according to the standard protocol. The quality of the RNA, fragmented RNA and cDNA libraries was assayed with an Agilent 2100 Bioanalyzer system (Agilent).
identifiers.namespace: Federal Research and Clinical Centre of Physical-C
identifiers.id: mycoplasma heatshock