Illumina HiSeq 2000 paired end sequencing; Distinctive responses of Mycobacterium smegmatis to exposure of low and high levels of hydrogen peroxide
Distinctive responses of Mycobacterium smegmatis to exposure of low and high levels of hydrogen peroxide
Log phase cultures (OD600 of 0.8 - 1.0) of M. smegmatis mc2155 were diluted 1:100 into 7H9 media and cultured for approximately 12 hours until the OD600 reached 0.3. Log phase cultures (OD600 of 0.8 - 1.0) of M. smegmatis mc2155 were diluted 1:100 into 7H9 media and cultured for approximately 12 hours until the OD600 reached 0.3. Re-inoculated cells were then treated with the indicated concentrations of H2O2 (0, 0.2 and 7 mM) for periods of 30 min or 3 h, and surviving cells were grown on 7H10 media. Cells were collected after 30 min of 0.2 mM or 7 mM H2O2 treatment, and the corresponding total RNAs were isolated and compared by RNA-sequencing to RNA from untreated cells that were prepared simultaneously. Briefly, total RNA of the treated mycobacteria mc2155 strains was treated using the RibominusTM Transcriptome Isolation Kit to remove rRNA contaminations. NEXTflexTM RNA Fragmentation Buffer was added for separating the mRNA into short fragments. Using these short fragments as templates, random hexamer-primers were used to synthesize the first strand of cDNA. The second strand of cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I. The short fragments were purified with the QIAQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). The short fragments were subsequently connected with sequencing adaptors.
Experimental Factor: 7mM hydrogen peroxide
stimulus