|
| Status |
Public on Aug 30, 2021 |
| Title |
RNA-seq of P. aeruginosa CZA-adapted isolates, sample: 2A5_B |
| Sample type |
SRA |
| |
|
| Source name |
Liquid culture (LB broth)
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
strain: MPAO1
genotype: mutSTn
treatment: Ceftazidime/avibactam
|
| Treatment protocol |
Bacterial cultures were serially passaged on increasing concentrations of Ceftazidime-avibactam prior to whole-genome sequencing and RNA-seq.
|
| Growth protocol |
Each isolate was streaked onto TSA with 5% Sheep Blood agar plates from glycerol stocks frozen at -80°C and incubated at 37°C for 20 hours. A quarter-loop of colonies was transferred and resuspended in fresh LB, in triplicate. Three biological replicates per isolate were included for the RNA-seq experiment. From these, 10mL liquid subcultures with a starting OD600 of 0.01 were grown in a vented 50mL tube at 37°C with shaking at 220 RPM for 12 hrs. At the mid-log growth phase (5 and 5.5 hours for the hypermutator and wild-type cells, respectively) 1.5mL of culture was mixed with 3mL of RNA-Protect Reagent (Qiagen, Germantown, MD, USA). Cell harvest was conducted as per the manufacturer’s instructions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with MagMAX Viral/Pathogen kit, modified to include a DNAse incubation step. One microgram of RNA underwent rRNA depletion with the NEBNext rRNA depletion kit for bacteria.
RNA-Seq libraries were prepared with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® and NEBNext Multiplex Oligos for Illumina on an epMotion 5075 instrument.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
11182020_PA_RNA_2A5_B_ADC_NX12_S16_S16
|
| Data processing |
Adapters were trimmed using Trimmomatic
Reads were mapped to reference using BWA v0.7.17 using default parameters
Samtools v1.11 and Picard CollectRnaSeqMetrics (Picard tools v2.25.0) were used to obtain QC metrics.
Bam alignments from 10 resequenced libraries were merged using samtools. All Bam files and the PAO1 reference GFF file (Available from https://pseudomonas.com/downloads/pseudomonas/pgd_r_20_2/Pseudomonas_aeruginosa_PAO1_107/Pseudomonas_aeruginosa_PAO1_107.gff.gz , accessed 01/18/2021) were used to generate read counts for each sample with HTseq v0.11.4, using the htseq-count -s reverse -m union flags
Genome_build: GCA_000006765.1
Supplementary_files_format_and_content: htseq-count tabular format (.tsv)
|
| |
|
| Submission date |
Jul 14, 2021 |
| Last update date |
Aug 30, 2021 |
| Contact name |
Augusto Dulanto Chiang |
| E-mail(s) |
dulantoa@nih.gov
|
| Organization name |
NIAID
|
| Street address |
9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20814 |
| Country |
USA |
| |
|
| Platform ID |
GPL28386 |
| Series (1) |
| GSE180086 |
Novel Mechanisms of Antibiotic Resistance in Mismatch Repair-Deficient Pseudomonas aeruginosa hypermutators |
|
| Relations |
| BioSample |
SAMN19595008 |
| SRA |
SRX11087361 |
