|
| Status |
Public on Jun 14, 2014 |
| Title |
DirPhiKZ_15min_2 |
| Sample type |
SRA |
| |
|
| Source name |
Cells infected with PhiKZ phage
|
| Organism |
Phikzvirus phiKZ |
| Characteristics |
infection: Infected
host strain: PAO1 Krilov
stage of infection: Middle Infection
directional reads: Directional
|
| Growth protocol |
Bacteriophage PhiKZ was amplified from confluent lysed agar plates, purified by 0.45µm filtration and concentrated after incubation in 10% PEG8000/1M NaCl according to standard procedures (Ceyssens et al., 2006). P. aeruginosa and its derivative strains were grown overnight in 5 ml LB medium to reach saturation. Cells were diluted 1:100 in 2-500 mL fresh medium, grown at 37°C and, in infection experiments, infected with MOI 5 at OD600 nm of 0.3. Cell growth (or phage infection) was halted at indicated time points by rapid cooling in ice water or by methods specific for RNA-seq samping (see below). Cells were harvested by centrifugation (6,000 x g), flash-frozen and stored at -80°C until use. The efficiency of infection was always checked by cell counts of the infected culture five minutes post infection, which should contain <5% surviving cells compared to the non-infected culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells intended for RNA sequencing were rapidly suspended in 1/10th volumes of ice cold stop solution (10% phenol in ethanol) and chilled to inhibit RNA transcription and degradation at indicated time points. Cells were then collected by centrifugation (6,000 x g) and Total RNA was extracted from the resulting cell pellet by vigorously resuspending it in TRIzol (Ambion) and performing a classical phenol chloroform extraction followed by ethanol precipitation. Remaining DNA was removed using TURBO DNAse (Ambion), the efficiency of which was checked by the absence of reporter PCR products of both bacterial and phage genes.
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Directional reads appropriate for the construction of operon maps
|
| Data processing |
Sequenced reads were trimmed for adaptor sequence, and then mapped to either the PhiKZ phage (AF399011.1) or P. aeruginosa whole genome using CLC Genomics Workbench 7.0.3 with strand specific alignment in some samples.
The comparisons of relative levels of expression for gene features between different time point after infection within both the phage and host genomes were made with the negative binomial distribution test using the DESeq Bioconductor package in R.
Genome_build: PhiKZ phage (AF399011.1) or P. aeruginosa whole genome
Supplementary_files_format_and_content: Tab-delimited text files include RPKM and TGR values for each gene feature of the phage or host for each sample. Additionally, two supplementary tab-delimited text files are included with relative expression values between timepoints calculated using the DESec Bioconductor package binomial distribution test in R for the gene features of either the phage or host.
|
| |
|
| Submission date |
Jun 13, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Bob Gordon Blasdel |
| E-mail(s) |
blasdelb@gmail.com
|
| Phone |
+32 488 08 36 83
|
| Organization name |
KU Leuven
|
| Department |
Afdeling Gentechnologie
|
| Lab |
Laboratory of Gene Technology
|
| Street address |
Kasteelpark Arenberg 21, bus 2462
|
| City |
Leuven |
| State/province |
Vlaams-Brabant |
| ZIP/Postal code |
3001 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL18804 |
| Series (1) |
| GSE58494 |
RNA-Seq analysis of Pseudomonas aeruginosa PAO1 cells infected with PhiKZ phage |
|
| Relations |
| BioSample |
SAMN02854662 |
| SRA |
SRX597266 |
