NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM5452586 Query DataSets for GSM5452586
Status Public on Aug 30, 2021
Title RNA-seq of P. aeruginosa CZA-adapted isolates, sample: 2B4_A
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_2B4_A_ADC_NX12_S25_S25
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 SAMN19595019
SRA SRX11087405

Supplementary file Size Download File type/resource
GSM5452586_11182020_PA_RNA_2B4_A_htsq.tsv.gz 22.6 Kb (ftp)(http) TSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap