|
| Status |
Public on Sep 26, 2023 |
| Title |
biol rep 2, S. aureus + S. anginosus, control, 1h |
| Sample type |
SRA |
| |
|
| Source name |
HTEpiC- Human tonsils epithelial cells
|
| Organism |
Staphylococcus aureus |
| Characteristics |
cell line: HTEpiC- Human tonsils epithelial cells
cell type: primary epithelial cell line
time: 1h
treatment: bacterial mix without Host
group: ctr_1h.BR1
|
| Growth protocol |
The bacterial strains grown to log phase OD600nm =0.8 -1.2 were diluted to OD600nm = 0.36 – 0.42 (corresponding to 0.6 - 1.2 x 108 CFU/ml) and seeded into wells with host cells at a Multiplicity of Infection (MOI) of 5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 1 and 3h post-infection, the media was aspirated, and the host cells were washed twice with fresh media to remove unbound bacteria. The host cells were then trypsinized and lysed with Triton-X. The released bacterial mix was then collected from three technical replicates and pooled together for RNA extraction. Plate enumeration, adhesion assay and cytotoxicity assay were also performed.
Total RNA extracted from three replicates of S. aureus cocultured with S. anginosus in the absence/presence of host cells collected at time points of 1 h and 3 h were processed for RNA-seq library preparation. The samples were sequenced on an Illumina 550 platform, with dual indexes, and paired-end mode. The final sequencing concentration was 1.8 pM.
Prior to RNA libraries for RNA-seq, depletion of bacterial rRNA was performed with the RiboCop rRNA depletion kit for Gram-Positive Bacteria (G+), according to the manufacturer’s protocol. In total 12 samples were processed for libraries construction using Lexogen’s CORALLTM Total RNA-Seq Kit with RiboCop following the manufacturer's protocols.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
BR2.reads aligned against S. aureus from bacterial mix without host at 1h
|
| Data processing |
RNA seq reads were generated from 12 samples in two runs of RNA-seq. The overall pipeline for RNA-seq data analysis included generating FASTQ-format files containing reads sequenced from an NGS platform, quality control and trimming, aligning reads to an annotated reference genome, and quantifying expression of genes.
Each library produced between 45 – 184 million reads, were pre-processed for quality check using FASTQC/0.11.9-Java-11. Filtering (removable of adaptor dimer reads) and trimming (removable of low-quality bases) was performed by Trimmomatic/0.39-Java-11. Then, only those sequences with a quality score Q > equal to 20 and a minimum of 55 nucleotide sequence lengths were retained in the dataset.
The final quality check was performed in the trimmed file. S. aureus TR145 strain was used as a reference genome for the mapping performed by Bowtie2/2.4.4-GCC-10.3.0. For all the samples, percentage of mapping efficacy was retrieved from Bowtie2 and sorted by Samtools/1.14-GCC-11.2.0.
Further, the gene count matrix for gene expression analysis was identified using the featureCounts program implemented in the SourceForge Subread package.
Differentially expressed genes analysis of the test group and the control group (each group has three biological replicates per conditions) was analyzed using the DESeq R package (1.38.0).
DEGs were calculated under log2|FoldChnage|(log2|FC|) > 2 and a false discovery rate (FDR) adjusted p (padj) < 0.05.
Supplementary files format and content: raw RNA seq reads generated after RNA sequencing for each Samples (total 12)
Supplementary files format and content: tab-delimited text files include count matrix complied after feature count tool for each Sample
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| |
|
| Submission date |
Jun 14, 2023 |
| Last update date |
Sep 26, 2023 |
| Contact name |
Anne Merethe Hanssen |
| E-mail(s) |
anne-merethe.hanssen@uit.no
|
| Organization name |
University of Tromsø
|
| Department |
Medical Biology
|
| Lab |
Host microbe interaction
|
| Street address |
Hansine Hansens veg 18
|
| City |
Tromsø |
| ZIP/Postal code |
9019 |
| Country |
Norway |
| |
|
| Platform ID |
GPL28116 |
| Series (1) |
| GSE234900 |
Coculturing with Streptococcus anginosus alters Staphylococcus aureus transcriptome when exposed to tonsillar cells |
|
| Relations |
| BioSample |
SAMN35732228 |
| SRA |
SRX20676058 |
