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Sample GSM1890775 Query DataSets for GSM1890775
Status Public on Apr 06, 2016
Title FPR13C
Sample type SRA
 
Source name Bacteria
Organism Pseudomonas aeruginosa
Characteristics strain: PAO1
genotype: wild type
incubation time: 13 hours incubation after the standard inoculation
fna level: 0
Treatment protocol When the cultures were in early log phase of growth, eleven hours after inoculation, FNA was added with starting concentration of 0.1 mg N/L. Control cultures (no added FNA) had the same volume of sterilized Milli-Q water added. Experiments of the control and FNA treated cultures were conducted in triplicates. Samples were taken for RNA extraction from the triplicate FNA treated and the triplicate control cultures of PAO1 after 13 hours incubation, that is 2 hours after FNA addition.
Growth protocol PAO1 was activated according to supplier's instructions and grown on tryptic soy agar plates at 30 ºC for 26 hours. One colony was then transferred to tryptic soy broth medium and incubated shaken at 150 rpm and 30 ºC for another 26 hours. Afterwards, 5 mL of the bacterium suspension was transferred into serum bottles containing 150 mL of sterile anaerobic GLYM9 medium in an anaerobic chamber. After 26 hours’ incubation the optical density at 600 nm (OD600) of the culture was adjusted to 0.5. Then 10 mL of this culture was transferred into 150 mL of sterile anaerobic GLYM9 medium in serum bottles in an anaerobic chamber and then incubated at 30 ˚C while shaken at 150 rpm.
Extracted molecule total RNA
Extraction protocol 5 mL of the bacteria suspension from each serum bottle was centrifuged at 13,000 x g for 2 minutes, the supernatant was discarded and the pellets were immediately frozen in the liquid nitrogen before storing at -80ºC for later RNA extraction. Total RNA extraction was performed using the QIAGEN miRNeasy Mini Kit (Catalog number: 217004) according to the manufacturer’s instructions except adding an extra bead-beating step to ensure the complete lysis of the bacterial cells.
Strand-specific cDNA library construction
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Data processing NGS QC Toolkit (v2.3.3) was used to do the quality control and tag removal to generate the clean sequence reads; the resulting clean reads no shorter than 75 bp were used for downstream analyses.
The cleaned sequence reads for each sample were aligned to the PAO1 reference genome (NC_002516) using SeqAlto
Strand-specific coverage for each gene was calculated and differential expression analysis was conducted using the cuffdiff command in Cufflinks (version 2.2.1) on triplicated cultures
Statistic analyses and visualization were conducted using the cummeRbund package in R (http://compbio.mit.edu/cummeRbund/). Gene expression was calculated as reads per kilobase of a gene per million mapped reads (RPKM), a normalized value derived from the frequency of detection and the length of a given gene (Cole Trapnell, Nature Protocols, 2012) Differences in fold-change values were calculated between control and FNA-treated samples (0.1 mg N/L) by determining the log2 fold-change (LFC) of the averaged RPKM values determined from triplicate experiments run on two separate occasions.
Genome_build: NC_002516
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...
 
Submission date Sep 22, 2015
Last update date May 15, 2019
Contact name Shuhong Gao
E-mail(s) gaoshuhong1127@163.com
Phone +61 (0) 7 334 67217
Organization name The University of Queensland
Department Advanced Water Management Centre
Lab Gehrmann Laboratories (60#)
Street address Research Road, St Lucia
City Brisbane
State/province QLD
ZIP/Postal code 4072
Country Australia
 
Platform ID GPL18644
Series (1)
GSE73323 Next generation sequencing of Pseudomonas aruginosa PAO1 in the presence of Free Nitrous Acid stress
Relations
BioSample SAMN04101978
SRA SRX1273292

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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