|
| Status |
Public on Nov 13, 2020 |
| Title |
infected_A+TEX |
| Sample type |
SRA |
| |
|
| Source name |
infected samples (TEX treated)
|
| Organisms |
Phikzvirus phiKZ; Pseudomonas aeruginosa PAO1 |
| Characteristics |
infection status: infected with phiKZ
strain: PA01
|
| Treatment protocol |
P. aeruginosa was grown to early exponetial phase before being infected with phiKZ (MOI of 15) for 10min. Uninfected bacteria were used as control.
|
| Growth protocol |
P. aeruginosa was grown in lysogeny broth (LB) at 37°C and 220rpm. Bacteriophage phiKZ was expanded by liquid culture and purified by subsequent filitration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Flash frozen samples were used for RNA using the Hot phenol protocol (described e.g. in PMID: 31796567)
Total RNA extracted from ɸKZ-infected and uninfected P. aeruginosa PAO1 cells was divided in two pools, named TEX- and TEX+. Samples were examined by capillary electrophoresis and fragmented using ultrasound (4 pulses of 30 sec at 4 °C), followed by T4 Polynucleotide Kinase (NEB) treatment. TEX+ samples were subjected to Terminator-5´-phosphate-dependent exonuclease to enrich primary transcripts whereas TEX- samples remained untreated. For cDNA synthesis RNA samples were poly(A)-tailed and remaining 5´PPP structures were removed. After ligation of an RNA adapter to the 5´P end, first-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. PCR amplification of cDNAs was conducted using a high-fidelity DNA polymerase and cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). Quality control was carried out by capillary electrophoresis. All described procedures were performed by Vertis Biotechnology (Germany).
TruSeq Stranded Library Preparation Kit (Illumina)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
infected samples (TEX treated)
|
| Data processing |
Demultiplexing
FastQ quality trimming using FastX and a cut-off value of 20
Fastq to fasta conversion using FastX (v0.11.7)
Size filtering: discarding reads shorter than 20 nt (via READemption v0.4.3)
Read mapping via READemption (v0.4.3)
Counting of reads per gene via READemption (v0.4.3)
Genome_build: Pseudomonas aeruginosa (AE004091.2); Pseudomonas virus phiKZ (AF399011.1)
Supplementary_files_format_and_content: CSV (comma separated) of gene count per gene per sample
|
| |
|
| Submission date |
Jun 23, 2020 |
| Last update date |
Nov 13, 2020 |
| Contact name |
Falk Ponath |
| E-mail(s) |
Falk.Ponath@helmholtz-hiri.de
|
| Organization name |
Helmholtz Institute for RNA-based Infection Research (HIRI)
|
| Street address |
Josef-Schneider-Straße 2
|
| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL28760 |
| Series (1) |
| GSE153067 |
Introducing Differential RNAseq mapping to track the phage infection process for Pseudomonas virus ɸKZ |
|
| Relations |
| BioSample |
SAMN15351856 |
| SRA |
SRX8603012 |
