Illumina MiSeq paired end sequencing; RNA-seq of Azotobacter vinelandii cultured in with or without a nitrogen source at 5%, 10%, or 20% oxygen concentrations
RNA-seq of Azotobacter vinelandii cultured in with or without a nitrogen source at 5%, 10%, or 20% oxygen concentrations
WT_wN_10% O2_rep3_p
The cells were harvested at 3,000 × g for 2 min at room temperature, and washed by ice-cold phosphate solution (0.1 g K2HPO4 and 0.4 g KH2PO4 in 450 mL distilled water) Wild type A. vinelandii strains were cultured on MB plates with a nitrogen source for 2 days, inoculated into MB liquid medium without a nitrogen source with an initial OD600 of 0.1, and incubated for 24 h at 30°C and 300 rpm. The cells were harvested and inoculated into MB liquid medium with or without a nitrogen source with an initial OD600 of 0.5. The culture vials were purged with N2 for 1 min and sealed, and then O2 was injected into the air layer to bring O2 concentration to 5%, 10%, or 20%. After 2 h incubation, the cells were collected. Total RNA was extracted from each culture with TRIzol reagent (Invitrogen, NY, USA). RNA integrity was evaluated for quality control with an Agilent Bioanalyzer 2,100 using Agilent RNA 6,000 Nano Kit (Agilent technologies, CA, USA). After the quality evaluation, rRNAs were removed with RiboMinus Transcriptome Isolation Kit, bacteria (Thermo Fisher Scientific, MA, USA). cDNA library preparation was performed using KAPA RNA HyperPrep Kit Illumina Platforms (Kapa Biosystems, MA, USA). Prepared cDNA libraries were validated with an Agilent Bioanalyzer 2,100 using an Agilent High Sensitivity DNA Kit (Agilent Technologies).