|
| Status |
Public on Mar 25, 2020 |
| Title |
Control replicate 3 |
| Sample type |
SRA |
| |
|
| Source name |
cultured cells
|
| Organism |
Staphylococcus aureus |
| Characteristics |
strain: SH1000
|
| Growth protocol |
For differential expression analysis, bacterial reference samples (hereafter named “control samples” in RNA-seq experiments) were collected from an overnight culture (MHB-CA supplemented with 125 ng/mL tetracycline) by centrifugation at 5000g for 5 min, resuspended in RPMI 1640 and incubated for 30 min at 37°C. This bacterial suspension was then mixed with a cell lysate obtained from non-infected macrophages incubated in RPMI 1640 with 10 % FBS for 24 h. The relative amount of bacteria and cells was adjusted to that obtained in 24 h-infected cells. Intracellular bacteria were collected from macrophages as described for flow cytometry. Bacteria were sorted on the basis of their propidium iodide profile and GFP expression level using the gating methods described above, in a FACSAria III cytometer operated by the BD FACSDiva 8.0.1 software (BD Biosciences).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
S. aureus recovered from infected macrophages or from control samples were Centrifuged at 5000g for 5 min and lysed using the following procedure: pellets were resuspended in Tris-EDTA buffer containing freshly prepared 13 mg/mL lysozyme (Sigma) and 130 μg/mL lysostaphin (Sigma) for 30 min at room temperature. Resulting suspensions were processed for total RNA extraction with RNA extraction InviTrap Spin Universal RNA Mini Kit (Stratec) following the manufacturer’s instructions. Traces of contaminating genomic DNA were removed from samples by treatment with TURBO DNase (Ambion) for 30 min at 37°C according to the manufacturer’s instructions. RNA purity was checked using a NanoDropM spectrophotometer (Thermo Fisher Scientific).
Total RNA from three independent replicates were checked on the Bioanalyser system (Agilent) for its quality and integrity. Ribosomal RNA depletion was performed using the Bacteria RiboZero kit (Illumina). From rRNA-depleted RNA, directional libraries were prepared using the TruSeq Stranded mRNA Sample preparation kit following the manufacturer’s instructions (Illumina). Libraries were checked for quality on Bioanalyser DNA chips Bioanalyser (Agilent). Quantification was performed with the fluorescent-based quantitation Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Sequencing was performed as an SRM run (SR: Single Read, PE: Paired-end Reads, M: multiplexed samples) for 65 bp sequences on HiSeq 2500 Illumina sequencer (65 cycles). The multiplexing level was 6 samples per lane.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ctrl6
|
| Data processing |
Reads were cleaned of adapter sequences and low-quality sequences using cutadapt version 1.11. Only sequences at least 25 nt length were considered for further analysis.
Bowtie version 0.12.7 with default parameters, was used for alignment on the reference genome (CP000253.1, NCBI)
Genes were counted using featureCounts version 1.4.6-p3 from Subreads package (parameters: -t gene -g ID -s 1).
Count data were analyzed using R version 3.4.1 and the Bioconductor package DESeq2 version 1.16. The normalization and dispersion estimation were performed with DESeq2 using the default parameters and statistical tests for differential expression were performed applying the independent filtering algorithm. A generalized linear model was set in order to test for the differential expression between the intracellular persisters and control conditions. Raw P values were adjusted for multiple testing according to the Benjamini and Hochberg (BH) procedure and genes with an adjusted p value lower than 0.05 were considered differentially expressed.
Genome_build: CP000253.1 from NCBI
Supplementary_files_format_and_content: Matrix from DESseq2
|
| |
|
| Submission date |
Oct 31, 2019 |
| Last update date |
Mar 25, 2020 |
| Contact name |
Rachel Legendre |
| E-mail(s) |
rachel.legendre@pasteur.fr
|
| Organization name |
Institut Pasteur
|
| Department |
Research and Resource Center for Scientific Informatics
|
| Lab |
Hub of Bioinformatics and Biostatistics
|
| Street address |
28, rue du docteur Roux
|
| City |
Paris |
| ZIP/Postal code |
75724 |
| Country |
France |
| |
|
| Platform ID |
GPL19006 |
| Series (1) |
| GSE139659 |
Intracellular Staphylococcus aureus persisters upon antibiotic exposure |
|
| Relations |
| BioSample |
SAMN13168919 |
| SRA |
SRX7080617 |
