|
| Status |
Public on Mar 30, 2023 |
| Title |
biol rep 2, S.aureus+ tonsillar cells , test, 3h |
| 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: 3h
treatment: with HOST
group: T3_Rep2
|
| Treatment protocol |
HTEpiC was seeded at a density of ~ 4 × e-5 viable cells per well in six-well plates coated with PLL. S. aureus TR145 at OD600nm of 0.4 was added to HTEpiC monolayer or to empty wells (coated with PLL) in a number corresponding to Multiplicity of Infection (MOI)=5 and incubated for 1h and 3h of exposure.
|
| Growth protocol |
HTEpiC- Human tonsil epithelial cells were cultured using Tonsil Epithelial Cell Medium (TEpiCM, Sciencell) supplemented with 1% Tonsil Epithelial Cell Growth Supplement (TEpiCGS) and penicillin/streptomycin solution (P/S) at 37°C in a 5% CO2 incubator. Prior to the culturing of HTEpiC, the T-75 tissue culture flask was coated with 2 µg/cm2 poly-L-lysin (PLL) and incubated at 37°C for 2h or overnight.
|
| 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 bacteria were then collected from three technical replicates and pooled together for RNA extraction. Plate enumeration, adhesion assay and cytotoxicity assay were also performed.
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.2 ug of total RNA was used for the construction of sequencing libraries.
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 15 samples were processed for libraries construction using Lexogen’s CORALLTM Total RNA-Seq Kit with RiboCop following he manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
RNA seq reads were generated from 14 samples in two runs of RNA-seq. Out fo these only 11 samples were processed for DEseq2 analysis.
Sequence reads were quality checked and trimmed for adaptor sequence/low-quality sequence using FASTQC (0.11.9-Java-11 ) and Trimmomatic (0.39-Java-11) respectively.Only those sequences with the quality score Q > equal to 20 and a minimum of 55 nucleotide sequence length were retained in the dataset.
Trimmed sequence reads were mapped to S. aureus TR145 throat isolate using Bowtie2 (2.4.4-GCC-10.3.0)
Reads mapped to each gene were identified using the HTSeq (v0.11.1) counting tool. After aligning reads to a reference and generating count files, it was further analyzed by DESeq2 (Bioconductor package) on the R platform to explore any DEGs present in the sample.
Deseq Normalization was performed followed by visualization of sample variance using principal component analysis (PCA). S. aureus only at 0h was not used for DESeq2 analysis. It was visualized only from PCA plot.
The DE was further curated to give only those genes which show p-value adjusted (padj) along the indication of the gene name. The threshold for padj was adjusted to less than 0.05 (padj < 0,05) and for log2foldchange greater than 2 (LFC> 2).
Assembly: GCA_949730015
Supplementary files format and content: raw RNA seq reads generated after RNA sequencing for each Samples
Supplementary files format and content: tab-delimited text files include count matrix complied after HTseq counting tool for each Sample
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| |
|
| Submission date |
Feb 28, 2023 |
| Last update date |
Mar 30, 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) |
| GSE226317 |
Exploring differentially expressed genes of staphylococcus aureus exposed to human tonsillar cells using RNA sequencing |
|
| Relations |
| BioSample |
SAMN33535712 |
| SRA |
SRX19531031 |
