title: GSM8615007: SL1344, PolB, rep1; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344; RNA-Seq
library_construction_protocol: S.typhimurium and its CpxA-delated strain were inoculated into LB broth containing 0.125 μg/ml polymyxin B, and cultured overnight at 37°C, followed by dilution and subculture. After growing to the logarithmic phase, the bacteria were washed twice with PBS, collected, quickly frozen in liquid nitrogen, and then sent to Beijing Novogene Bioinformatics Technology Co., Ltd. for transcriptome sequencing and analysis. 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.
identifiers.namespace: GEO
identifiers.id: GSM8615007_r1