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Sample GSM5982435

Query DataSets for GSM5982435

Status

Public on Nov 29, 2022

Title

wD3H4

Sample type

SRA

Source name

Bacterial cells

Organism

Staphylococcus aureus

Characteristics

strain: USA300
genotype: Wildtype
growth medium: TSB
growth conditions: D3 Healthy BAL, 12 h, 37 C

Treatment protocol

Mice were intranasally (IN) administered with saline or influenza A/PR/8/34 (PR8) and were left to recover for 3, 7, 10, 21 days post-IN (D3, D7, D10, D21). After each timepoint, mice were sacrificed and whole lungs were washed 3 times with 1mL cold PBS. Bronchoalveolar lavage (BAL) fluid was centrifuged and the supernatant, the cell-free portion, was used for bacterial culturing. Overnight WT MRSA cultures were cultured in saline BAL (wD3H, wD7H, wD10H, wD21H) and PR8 BAL (wD3P, wD7P, wD10P, wD21P) for 12 hours at 37°C, with 1000rpm of horizontal shaking. Overnight SaePQRS mutant MRSA cultures were cultured in D7 and D10 IN PR8 BAL (dD7P and dD10P) for 12 hours at 37°C, with 1000rpm of horizontal shaking. All bacterial samples were centrifuged and then the pellet was resuspended in PBS. The opitical density (OD) of WT and SaePQRS mutant bacterial suspensions were measured and diluted to an OD of 2. DIluted bacteria was centrifuged for pellets were frozen.

Growth protocol

USA300 (wildtype, WT, and SaePQRS mutant) were cultured in TSB from frozen stock for 18-24 hours. Overnight WT MRSA cultures were diluted 1:1000 in 40% (v/v) cell-free BAL for 12 hours at 37°C, with 1000rpm of horizontal shaking.

Extracted molecule

total RNA

Extraction protocol

Frozen bacterial pellets were resuspended in TE buffer, bead beat, and then RNA isolated using Qiagen RNeasy Mini Kit (Cat#74104). Contaminating DNA was removed by treatment with TURBO DNA-free kit (Cat#AM1907).
RNA libraries were prepared using Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit (Cat#20040529) with 1 ug of total RNA for the construction of sequencing libraries.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Description

Gene expression data from bacteria cultured in D3 healthy BAL for 12 hours

Data processing

Illumina reads were trimmed for adaptor sequence and for quality in CLC Genomics Workbench (v.21.0.5)
Trimmed sequences were mapped to to the reference genome sequence of Staphylococcus aureus USA300, with the parameters: mismatch cost, 2; insertion and deletion cost, 3; length and similarity fraction, 0.8.
Uniquely mapped transcripts from each sample were exported from CLC Genomics Workbench and used for normalization and differential gene expression analysis with an R package, DESeq2 version 1.32.0
Assembly: USA300_FPR3757
Supplementary files format and content: MRSA_gene_counts_matrix contains uniquely mapped transcripts for each sample with gene name, length, USA300 new locus tag, USA300 old locus tag, and description.
Supplementary files format and content: D3_gene_exp_diff, D7_gene_exp_diff, D10_gene_exp_diff, and D21_gene_exp_diff contains differential gene expression between MRSA conditioned in healthy and PR8 BAL at each mouse timepoint (D3, D7, D10, and D21, respectively) post-IN administration. Files include gene names, baseMean, log2FoldChange, lfcSE, stat, pvalue, padj.
Supplementary files format and content: D7_TCS_gene_exp_diff and D10_TCS_gene_exp_diff, contains differential gene expression between WT and saePQRS mutant MRSA conditioned in PR8 BAL at each mouse timepoint (D7 and D10 respectively) post-IN administration. Files include gene names, baseMean, log2FoldChange, lfcSE, stat, pvalue, padj.

Submission date

Mar 29, 2022

Last update date

Nov 29, 2022

Contact name

Christophe Langouët-Astrié

E-mail(s)

christophe.langouetastrie@cuanschutz.edu

Organization name

University of Colorado Denver, Anschutz

Department

Pulmonary Sciences and Critical Care