|
| Status |
Public on Apr 17, 2021 |
| Title |
SBW25_pQBR57_d4242_AR04 |
| Sample type |
SRA |
| |
|
| Source name |
Pseudomonas fluorescens SBW25
|
| Organism |
Pseudomonas [fluorescens] SBW25 |
| Characteristics |
taxid: 216595
amelioration/genotype: dPFLU4242
plasmid: pQBR57
|
| Treatment protocol |
1 ml samples were added to 0.4 volumes of ice-cold 95% ethanol 5% phenol 'stop solution' [@Colgan2016-mu], mixed gently, and incubated on ice for at least 30 minutes. Each of the three replicates was processed separately as a block.
|
| Growth protocol |
Overnight cultures of bacteria were subcultured 1:50 into 6 ml prewarmed KB microcosms and grown at 28°C shaking at 180 rpm. Samples were harvested when cultures reached intermediate exponential phase (OD600 = 0.6, as assessed on a Tecan Spark plate reader).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were harvested at 4.5 G for 10 minutes, resuspended in 1 ml TRI reagent, and incubated at room temperate for 5 minutes. Amylene-stabilised chloroform (400 µl, Sigma-Aldrich 34854-M) was added to each tube, and incubated for 2-15 minutes at room temperature. Samples were then transferred to 5Prime Phase Lock Gel 'Heavy' tubes (VWR 733-2478) and centrifuged at 17 G for 15 minutes at room temperature. The aqueous phase was collected into a fresh microfuge tube and 450 µl isopropanol was added to precipitate RNA. Samples were incubated at room temperature for 30 minutes before pelleting at 17 G for 30 minutes. The pellet was rinsed by gently adding 1 ml of 70% ethanol and pelleting at 7.5 G for 5 minutes. The supernatant was carefully removed and the pellet allowed to air dry before resuspension in RNase-free water at 65°C. To remove any residual contaminating DNA, RNA was diluted to ≤200 ng/µl and treated with TURBO DNA-free RNase-free DNase (Invitrogen AM1907) according to the manufacturer's instructions. Reaction products were purified with Agencourt RNAClean XP beads. Ribosomal RNA was removed using the Bacterial RiboZero rRNA depletion kit (Illumina).
NEBNext Ultra-Directional RNA library preparation kit (Illumina). Sequencing is 'reverse-stranded'.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Sample 4
CountsMatrix.csv
de_table_chr_vanc.csv
de_table_comp.csv
de_table_pQ57.csv
rpkm_chr.csv
rpkm_pQ57.csv
4-AR04
|
| Data processing |
Trimmed using Cutadapt version 1.2.1 using the option -O 3
Trimmed using Sickle version 1.200 with a minimum window quality score of 20 and reads longer than 19 bp
Strand-specific alignment to the reference sequence(s) using HISAT 2.1.0 with options phred33, no-spliced-alignment, new-summary, and rna-strandness RF
Filtering using samtools to remove mapping with PHRED-scaled quality <10
Function featureCounts from Rsubread 1.30.9
Analysis using edgeR 3.22.5
Genome_build: Aligned to reference sequences AM181176, LN713926, AM235768
Supplementary_files_format_and_content: CountsMatrix.csv: raw counts for each feature for each sample. Output from Rsubread
Supplementary_files_format_and_content: CountsMatrix_stats.csv: statistics of the raw counts for each sample. Output from Rsubread
Supplementary_files_format_and_content: de_table_chr_vanc.csv: differential expression of chromosomal genes for each sample relative to the control. Output from edgeR
Supplementary_files_format_and_content: de_table_chr_comp.csv: differential expression of chromosomal genes for each sample relative to corresponding plasmid-bearing strains. Output from edgeR
Supplementary_files_format_and_content: de_table_pQ57.csv: differential expression for relevant samples relative to the wild-type pQBR57-bearer. Output from edgeR
Supplementary_files_format_and_content: de_table_pQ103.csv: differential expression for relevant samples relative to the wild-type pQBR103-bearer. Output from edgeR
Supplementary_files_format_and_content: edgeR_NormalizationFactors.csv: normalization factors for each sample. Output from edgeR
Supplementary_files_format_and_content: rpkm_*.csv: calculated RPKM values. Output from edgeR
|
| |
|
| Submission date |
Jun 01, 2020 |
| Last update date |
Apr 17, 2021 |
| Contact name |
James Hall |
| E-mail(s) |
j.p.j.hall@liverpool.ac.uk
|
| Organization name |
University of Liverpool
|
| Street address |
Crown Street
|
| City |
Liverpool |
| ZIP/Postal code |
L69 7ZB |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL28611 |
| Series (1) |
| GSE151570 |
Convergent transcriptional responses to acquisition and amelioration of different conjugative plasmids |
|
| Relations |
| BioSample |
SAMN15075949 |
| SRA |
SRX8449838 |
