Illumina HiSeq 3000 sequencing; RNAseq of S. meliloti Rm1021 all ecf/anti-σ deletion mutants overexpressing heterologous ecf genes compared to an empty vector control strain
RNAseq of S. meliloti Rm1021 all ecf/anti-σ deletion mutants overexpressing heterologous ecf genes compared to an empty vector control strain
Rm1021 ECF/anti-σ-free strain derivatives were grown to an OD600 of 0.1-0.3 in MOPS buffered minimal medium with gentamicin (6 µg/mL). The OD600 was adjusted to 0.1 and IPTG was added at a final concentration of 500 µM. Strains were grown for ~ 8-9 hours until cell density reached an OD600 of 0.8 and collected by centrifugation at 10,000 rcf for 5 min. Cell pellets were frozen in liquid nitrogen and kept at -80 °C. Assays were performed in triplicates for each strain. For total RNA isolation, the Qiagen miRNeasy Kit was used starting from a pellet of 8 mL cultured cells. The cell pellet was resuspended in 1 mL QIAzol lysis reagent and homogenized in a FastPrep® sample preparation system using Lysing Matrix B containing 0,1 mm silica beads (MP biomedicals) and the following settings: 3x 6500 rpm for 20 sec, 15 sec break. The supernatant was transferred into a new tube and RNA isolation was performed according to the manufacturer's instruction including the optional on column DNaseI digestion. rRNA depletion was conducted with the Illumina Ribo-Zero rRNA removal Kit (Bacteria). The cDNA library was prepared using the NEB Ultra directional Kit.