title: Illumina MiSeq sequencing; RNA-Seq of sorted and unsorted prophage-induced subpopulations in Corynebacterium glutamicum
design: RNA-Seq of sorted and unsorted prophage-induced subpopulations in Corynebacterium glutamicum
library_construction_protocol: 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: Experimental Factor: stimulus
attributes.value: counter-silencing
attributes.tag: Experimental Factor: compound
attributes.value: isopropyl beta-D-thiogalactopyranoside
attributes.tag: Experimental Factor: dose
attributes.value: 150
attributes.tag: Experimental Factor: phenotype
attributes.value: negative
attributes.tag: Experimental Factor: protocol
attributes.value: 10^6 cells, RNAlater