title: Rm1021 ECF/anti-σ-free strain overexpressing ecf02_2817 rep1
organism: Sinorhizobium meliloti
description: Protocols: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-01-26
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-01-26
attributes.tag: External Id
attributes.value: SAMEA110702365
attributes.tag: INSDC center alias
attributes.value: Philipps-Universitat Marburg Center for Synthetic Microbiology (Synmikro) Becker lab
attributes.tag: INSDC center name
attributes.value: Philipps-Universitat Marburg Center for Synthetic Microbiology (Synmikro) Becker lab
attributes.tag: INSDC first public
attributes.value: 2023-01-26T00:16:52Z
attributes.tag: INSDC last update
attributes.value: 2023-01-26T00:16:52Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12126:Rm1021 ECF/anti-σ-free strain overexpressing ecf02_2817 rep1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: developmental stage
attributes.value: vegetative growth
attributes.tag: genotype
attributes.value: Rm1021 Δecf/anti-σ pSRKGm [m] Plac/T5 ecf02_2817
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-12126:Rm1021 ECF/anti-σ-free strain overexpressing ecf02_2817 rep1
attributes.tag: strain
attributes.value: Rm1021