|
| Status |
Public on Mar 13, 2024 |
| Title |
LUZ24_enriched |
| Sample type |
SRA |
| |
|
| Source name |
Pseudomonas aeruginosa Li010, Pseudomonas virus LUZ24
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
cell type: bacterial cell
strain: Li010
treatment: enriched for primary RNAs
agent: Pseudomonas virus LUZ24
infection stage: infected with phage (samples from different timepoints during infection pooled)
|
| Treatment protocol |
Total RNA was extracted using hot-phenol extraction, followed by DNAse treatment. For each phage, the samples from different timepoints were pooled in equal amounts before starting the library prepartion. The enriched samples were subjected to an enrichment procedure based on streptavidin beads to enrich for primary transcripts. For the control samples the enrichment steps were omitted.
|
| Growth protocol |
P. aeruginosa cells (PAO1/PAK/Li010) were grown to exponential phase in LB medium and infected with the appropriate phage (LUZ19, LUZ24, YuA, PAK_P3, 14-1 or phiKZ). Culture samples were taken at multiple timepoints during infection. The number of samples and the timepoints were decided depending on the duration of the phage infection cycle.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All samples, aside from 0 min, were pooled together and homogenized (sample tφ). The resulting samples t0 (uninfected) and tφ (phage infected) were subjected to hot phenol treatment and ethanol precipitation to extract and purify the RNATotal RNA was extracted using hot-phenol extraction, followed by DNAse treatment.
Total RNA was enriched for primary transcripts using the ONT-cappable-seq enrichment protocol. In parallel, control samples were subjected to similar environmental conditions. The RNA was reverse transcribed, PCR amplified and barcoded accoriding to Oxford Nanopore Technology cDNA-PCR protocol (SQK-PCB109)
Total RNA was enriched for primary transcripts using the ONT-cappable-seq enrichment protocol. In parallel, control samples were subjected to similar environmental conditions. The RNA was reverse transcribed, PCR amplified and barcoded accoriding to Oxford Nanopore Technology cDNA-PCR protocol (SQK-PCB109). The amplified cDNA samples were pooled together in a final library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
| |
|
| Data processing |
Live basecalling and demultiplexing by promethION 24 device
Identification and orientation of full-length reads using Pychopper (v2.5.0)
Read trimming using Cutadapt (v2.7)
Mapping reads against reference genomes of P. aeruginosa host strain and phages using minimap2
Peak calling using termseq-peaks scripts
Assembly: PAO1 (NC_002516.2); PAK (LR657304.1); Li010 (CP124600); LUZ19 (NC_010326.1); LUZ24 (NC_010325.1); YuA (NC_010116.1); 14-1 (NC_011703.1); phiKZ (NC_004629.1); PAK_P3 (NC_022970.1)
Supplementary files format and content: Narrowpeak files generated by peak calling
|
| |
|
| Submission date |
May 04, 2023 |
| Last update date |
Mar 13, 2024 |
| Contact name |
Rob Lavigne |
| E-mail(s) |
rob.lavigne@kuleuven.be
|
| Organization name |
KU Leuven
|
| Department |
Biosystems
|
| Lab |
Laboratory of Gene Technology
|
| Street address |
Kasteelpark Arenberg 21
|
| City |
Leuven |
| ZIP/Postal code |
3001 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL31946 |
| Series (1) |
| GSE231702 |
Refining the transcriptional maps of Pseudomonas aeruginosa phages using ONT-cappable-seq |
|
| Relations |
| BioSample |
SAMN34591411 |
| SRA |
SRX20229489 |
