GSM6598763: WT_3; Actinobacillus pleuropneumoniae; RNA-Seq
The WT (S4074) and ∆cpxAR mutant strains were grown in TSB overnight, then transferred (at a 1:1000 dilution) into fresh medium and grown to an OD600 of 0.6 at 37°C or 42°C. Total RNA was extracted from the bacterial solution using a Bacteria Total RNA Isolation Kit (Sangon Biotech, China) following the manufacturer's protocol. Total RNA was used as input material for the RNA sample preparations. For prokaryotic samples, mRNA was purified from total RNA using probes to remove rRNA. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. And in the DNA polymerase I system, use dUTP to replace the dNTP of dTTP as the raw material to synthesize the second strand of cDNA. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. Then USER Enzyme was used to degrade the second strand of cDNA containing U, In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.