organism: Corynebacterium glutamicum
description: Protocols: Strain ATCC 13032, harvesting cells after cultivation and/or sorting using a FACS device. In general, counter-silencing covers mechanism that are used to overcome repressing activities of xenogeneic silencers like H-NS, Lsr2 or here CgpS. One way to achieve counter-silencing of CgpS activity is to use a truncated version of CgpS itself consisting only of the oligomerization domain (as described in Pfeifer et al., 2016, doi: 10.1093/nar/gkw692). Here we used 150 µM IPTG to overexpress the truncated version. For the counter-silencing experiments the expression of an genomically integrated eyfp was monitored by flow cytometry and sorted by FACS. The eyfp gene was cloned under a phage promoter called lys (for lysin), which can be activated by the counter-silencing mechanism (described below). For large volumes: RNeasy Kit (Qiagen, Hilden, Germany), cell disruption was performed with glass beads For small volumes (sorted samples): NucleoZol (Machery-Nagel, Düren, Germany) was used, cell disruption was performed with lysozyme + mutanlysin large sample volumes: rRNA depletion was done using Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria) (Epicentre/illumina, Munich, Germany) small volumes: a Protocol for Removal of rRNA from Small Amounts of Total RNA” from Clontech used All libraries were generated using: TruSeq Stranded emRNA Library Prep Kit (Illumina, Munich, Germany)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-09-15T17:01:47Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-05-29T15:57:37Z
attributes.tag: External Id
attributes.value: SAMEA4697073
attributes.tag: INSDC center name
attributes.value: IBG-1, Forschungszentrum Julich GmbH
attributes.tag: INSDC first public
attributes.value: 2018-09-15T17:01:47Z
attributes.tag: INSDC last update
attributes.value: 2018-05-29T15:57:37Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6809:Sample 21
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: IPTG-inducible expression of truncated CgpS; lys-EYFP
attributes.tag: phenotype
attributes.value: negative
attributes.tag: replicate
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-6809:Sample 21
attributes.tag: scientific_name
attributes.value: Corynebacterium glutamicum
attributes.tag: stimulus
attributes.value: counter-silencing
attributes.tag: strain
attributes.value: ATCC 13032
attributes.tag: type-material
attributes.value: type strain of Corynebacterium glutamicum