|
| Status |
Public on Jan 19, 2021 |
| Title |
RNA PAO1 ∆alpA + cipro replicate C |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cells grown to OD ∼0.3 to 0.5
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
genotype: deletion of alpA
|
| Treatment protocol |
mid-log cultures were treated with ciprofloxacin at a final concentration of 1 ug/mL for 120 minutes
|
| Growth protocol |
overnight cultures were back-diluted to OD600 of 0.01 in 3 mL LB and grown to mid-log-phase (OD600 of ~0.5)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted via Direct-Zol RNA Miniprep Kit (Zymo Research)
Illumina cDNA libraries were generated using a modified version of the RNAtag-Seq protocol. 500 ng - 1 µg total RNA was fragmented, depleted of genomic DNA, dephosphorylated and ligated to DNA adapters carrying 5'-AN8-3' barcodes of known sequence with a 5' phosphate and a 3' blocking group. Barcoded RNAs were pooled and depleted of rRNA using the RiboZero rRNA depletion kit (EpiCentre). Pools of barcoded RNAs were converted to Illumina cDNA libraries in 2 main steps: (i) reverse transcription of the RNA using a primer designed to the constant region of the barcode adaptor with addition of an adapter to the 3' end of the cDNA by template switching using SMARTScribe (Clonetech); (ii) PCR amplification using primers whose 5' ends target the constant region of the 3' or 5' adaptors and whose 3' ends contain the full Illumina P5 or P7 sequences.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
JP_1128_C
|
| Data processing |
Sequencing reads from each pooled sample were demulitplexed based on their associated barcod sequence using custom scripts; allowing up to 1 mismatch in the barcode, provided this did not make assignemtn of the read to a different barcode possible. Barcode sequencese were subsequently trimmed from the first read as were terminal G's from the second read that may have been added by SMARTScirbe during template switching.
Reads were aligned to the PAO1 reference genome (NCBI RefSeq NC_002516) using BWA
Read counds were assigned to the genes and other genomic features using custom scripts
Differential expression analysis was conducted with DESeq2.
Genome_build: NC_002516.2
Supplementary_files_format_and_content: tab-delimited text file includes read counts assigned to each feature for each sample
|
| |
|
| Submission date |
Jun 15, 2020 |
| Last update date |
Jan 19, 2021 |
| Contact name |
Simon L Dove |
| E-mail(s) |
simon.dove@childrens.harvard.edu
|
| Organization name |
Boston Children's Hospital and Harvard Medical School
|
| Department |
Division of Infectious Diseases
|
| Lab |
Dove Laboratory
|
| Street address |
300 Longwood Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL23999 |
| Series (2) |
| GSE152480 |
Control of a programmed cell death pathway in Pseudomonas aeruginosa by a ppGpp-responsive antiterminator [RNAtag-seq] |
| GSE152485 |
Control of a programmed cell death pathway in Pseudomonas aeruginosa by a ppGpp-responsive antiterminator |
|
| Relations |
| BioSample |
SAMN15238182 |
| SRA |
SRX8548632 |
