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Sample GSM2666928

Query DataSets for GSM2666928

Status

Public on Dec 31, 2017

Title

C1_1A_L1

Sample type

SRA

Source name

Bacteria

Organism

Pseudomonas aeruginosa

Characteristics

strain: PAO1
genotype: wild type
incubation time: 13 hours incubation after the standard inoculation
nanoparticle level: 1 mg/L CuO NPs

Treatment protocol

Eleven hours after the inoculation when the culture was in its early exponential phase of growth (OD of 0.2~0.3), a volume of a stock CuO nanoparticles solution was added to reach the initial nanoparticle concentration of 0.1, 1, 10 and 50 mg/L. Control cultures, with no nanoparticle addition, had the same volume of GLYM9 medium added.

Growth protocol

P. aeruginosa PAO1 strain was activated according to the supplier (DSMZ) and grown on tryptic soy agar (TSA) plates at 30 ºC for 26 hours. One colony was then selected and transferred to tryptic soy broth (TSB) medium and incubated shaken at 150 rpm and 30 ºC for another 26 hours. Afterwards, 5 mL of the bacterium suspension was transferred into the serum bottles containing 150 mL of sterile anaerobic Glycerol modified M9 (GLYM9) medium in an anaerobic chamber. After 26 hours’ incubation, optical density at 600 nm (OD600) was measured and then adjusted to 0.5. Then 10 mL of this inoculum standard 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. GLYM9 medium used in this study contained 10 g/L glycerol, 0.2 g/L yeast extract, 0.2 M phosphate buffer, 9 mM NaCl, 38 mM NH4Cl, 2 mM MgSO4•3H2O, 100 μM CaCl2•2H2O and 30 mM NaNO3 with the pH adjusted to 6.3±0.

Extracted molecule

total RNA

Extraction protocol

The total RNA extraction was performed at 13 hours incubation, that is 2 hours after nanoparticle exposure. The bacteria suspension of 6 mL from each serum bottle was collected and centrifuged at 14,000 rpm for 5 minutes at 4 oC, 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 beat-beading 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

Description

1mg/L CuO NPs addition

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)
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 (4 μg-N/L) by determining the log2 fold-change (LFC) of the RPKM values determined from 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

Jun 13, 2017

Last update date

May 15, 2019

Contact name

Jianhua Guo

E-mail(s)

j.guo@awmc.uq.edu.au

Organization name

University of Queensland

Department

Australian Centre for Water and Environmental Biotechnology