|
| 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 |
