Comment[ArrayExpressAccession]	E-GEOD-46810
MAGE-TAB Version	1.1			
Public Release Date	2013-05-10
Investigation Title	Nucleotide-resolution, genome-wide, 5'-end maps of the transcriptome of Propionibacterium acnes (cultured with and without potassium downshift)			
Comment[Submitted Name]	Nucleotide-resolution, genome-wide, 5'-end maps of the transcriptome of Propionibacterium acnes (cultured with and without potassium downshift)
Experiment Description	The differential RNA-seq data contained within this entry is complemented by global RNA-seq and microarray data, which is also deposited in GEO. Duplicate cultures of Propionibacterium acnes strain KPA171202 were grown exponentially in batch culture under anaerobic growth. Samples were taken following subculture with and without potassium downshift (i.e. removal from medium). This produced 4 samples; 2 replicates x 2 conditions.  Aliquots of each of the 4 samples were then incubated with or without incubation TAP (tobacco acid pyrophosphatase) before library construction.  Thus, 8 libraries were analysed.  TAP treatment allows the cloning and sequencing of 5' ends that were originally triphosphorylated.			
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl		
Person Last Name	Romero A.	Lin	Romero	McDowall
Person First Name	David	Yu-fei	David	Kenneth
Person Mid Initials			A	J
Person Email	efy4dr@leeds.ac.uk			
Person Affiliation	University of Leeds			
Person Address	University of Leeds, Woodhouse lane, Leeds, West Yorkshire, United Kingdom			
Person Roles	submitter			
Protocol Name	P-GSE46810-4	P-GSE46810-1	P-GSE46810-3	P-GSE46810-2
Protocol Description	The sample source of a read was determined by the barcoding. The barcodes for minus and plus TAP treatment respectively are GATCAG and TTAGGC for Culture 1 without downshift; TAGCTT and TGACCA for Culture 1 with downshift; GGCTAC and ACAGTG for Culture 2 without downshift; CTTGTA and GCCAAT for Culture 2 with downshift. For each of the 8 libraries (2 cultures x 2 conditions x 2 treatments), we counted the number of times each nucleotide position was the first in a sequence read using a simple script (unpubl resource). The forward and reverse strands were processed seperately.	Potassium downshift (depletion from growth media) was performed by growing a 100 mL culture of P. acnes in HSM to an O.D.600nm of 1.0, after which the culture was separated into two equal halves and cells harvested by centrifugation. One half was washed using standard HSM, then used to inoculate 100 mL of fresh HSM and reincubated. The other half was processed in the same way, except the HSM lacked potassium (KH2PO4 and K2HPO4). After 1 h of reincubation, 12.5 mL of stop solution (95% [v/v] ethanol; 5% [v/v] phenol) was added to inhibit cell metabolism (Kime et al. 2008. In RNA Turnover in Bacteria, Archaea and Organelles, Vol 447 (ed. LE Maquat, CM Arraiano), pp. 215), and the cells were harvested by centrifugation. When necessary, cell pellets were stored frozen at -80C.	Cell pellets of P. acnes were resuspended in Kirby mix (Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich.), 100 M-5L 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 -20M-0C for 1 h, and then harvested by centrifugation (13,000 x g for 30 min at 4M-0C) . Nucleic acid pellets were washed twice with 700 M-5L 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 5M-bM-^@M-^Y-sequencing adaptor was ligated to transcripts prior to fragmentation, thereby allowing the 5M-bM-^@M-^Y ends of both long and short transcripts to be detected. Each of the 8 libraries was constructed using a different barcode.	Propionibacterium acnes strain KPA171202 (Bruggemann et al. 2004. Science 305: 671) was cultivated in an anaerobic workstation (MACS-MG-1000, Don Whitely Scientific) at 34M-0C under 80% [v/v] N2, 10% [v/v] CO2, and 10% [v/v] H2. All analyses were done using cells cultivated without shaking in 100 mL of modified Holland Synthetic Medium (HSM) (Holland et al. 1979. J Appl Bacteriol 47: 383-394). Inocula were prepared in two stages. First a single colony isolated from the surface of reinforced clostridial agar (Hirsch and Grinstead 1954. J Dairy Res 21: 101) was used to inoculate 10 mL of tryptone-yeast extract-glucose (TYG) broth (Kim and Naylor 1966. Appl Microbiol 14: 690). After growing to stationary phase, an aliquot was used to inoculate 100 mL of TYG broth to an O.D.600nm of 0.2. The culture was then incubated to an O.D.600nm of 1.0, after which cells were harvested by centrifugation (3,000 x g for 20 min) and washed by first resuspending in 10 mL of HSM (pre-warmed to 34oC) and then repeating the harvesting step. Finally, the cells were resuspended in 10 mL of pre-warmed HSM and an appropriate aliquot was used to inoculate 100 mL of pre-warmed HSM to an O.D.600nm of 0.2.
Protocol Type	normalization data transformation protocol	sample treatment protocol	nucleic acid library construction protocol	growth protocol
Experimental Factor Name	POTASSIUM DOWNSHIFT			
Experimental Factor Type	potassium downshift			
Comment[SecondaryAccession]	GSE46810			
Comment[GEOReleaseDate]	2013-05-10			
Comment[ArrayExpressSubmissionDate]	2013-05-09			
Comment[GEOLastUpdateDate]	2013-05-13			
Comment[AEExperimentType]	RNA-seq of coding RNA			
Comment[AdditionalFile:Data1]	GSE46810_After_TAP_treatment_-_FWD_strand_AVG_values.txt			
Comment[AdditionalFile:Data2]	GSE46810_After_TAP_treatment_-_RVS_strand_AVG_values.txt			
Comment[AdditionalFile:Data3]	GSE46810_Before_TAP_treatment_-_FWD_strand_AVG_values.txt			
Comment[AdditionalFile:Data4]	GSE46810_Before_TAP_treatment_-_RVS_strand_AVG_values.txt			
Comment[SecondaryAccession]	SRP022251			
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/SRR849179-SRR849186			
SDRF File	E-GEOD-46810.sdrf.txt