title: GSM1138617: Without downshift - Culture 2; Propionibacterium acnes KPA171202; RNA-Seq
library_construction_protocol: Cell pellets of P. acnes were resuspended in Kirby mix (Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich.), 100 µL per 1 O.D.600nm unit, and transferred to Lysing Matrix B tubes containing fine silica beads (MP Biomedical). Tubes were then placed in a high-speed benchtop homogenizer (Fastprep-24, MP Biomedical; set at 6.5 M/s). Cells were lysed by three cycles of homogenisation for 1 min with cooling between each cycle in an ice-water bath for 1 min. Lysates were extracted using an equal volume of acidic phenol: chloroform: isoamyl alcohol (50: 50: 1) and then chloroform: isoamyl alcohol (49: 1). Nucleic acid in the aqueous phase was precipitated by adding NaCl to 150 mM and 2.5 x volumes of 100% [v/v] ethanol, chilling at -20°C for 1 h, and then harvested by centrifugation (13,000 x g for 30 min at 4°C) . Nucleic acid pellets were washed twice with 700 µL of 70% [v/v] ethanol, air dried for 5 min and resuspended in RNase-free water. To remove contaminating DNA, samples were treated with DNase I using conditions described by the vendor (Ambion) and extracted with phenol: chloroform as described above. The concentration and integrity of RNA samples were determined using a NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific) and agarose gel electrophoresis (Kime et al. 2008. In RNA Turnover in Bacteria, Archaea and Organelles, Vol 447 (ed. LE Maquat, CM Arraiano), pp. 215), respectively. Samples were enriched for mRNA using MICROBExpress-Bacteria beads, as described by the manufacturer (Ambion), Libraries were constructed by vertis Biotechnologie AG, Germany  (www.vertis-biotech.com) as a service that included treating an aliquot of each RNA sample with TAP. The 5’-sequencing adaptor was ligated to transcripts prior to fragmentation, thereby allowing the 5’ ends of both long and short transcripts to be detected. Each of the 8 libraries was constructed using a different barcode.
attributes.tag: GEO Accession
attributes.value: GSM1138617