|
| Status |
Public on Mar 19, 2024 |
| Title |
PBS_DONOR6 |
| Sample type |
SRA |
| |
|
| Source name |
Colon
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Colon
genotype: WT
treatment: PBS
|
| Treatment protocol |
8 to 14-week-old mice were treated twice a day for 3 days with 2.5 mg vancomycin dissolved in 100µl drinking water or PBS. Feces collected from vancomycin-treated mice housed at the SPF barrier facility were immediately transferred into an anaerobic chamber. Feces were vortexed in sterile PBS, debris allowed to settle by gravity, and supernatant transferred to new tubes. Sealed tubes were then removed from the anaerobic chamber and orally administered to germ-free Swiss Webster mice via gavage. Inoculated mice were then transferred to Tecniplast IsoCages to prevent outside contamination.
|
| Growth protocol |
Mice grown under SPF or germ-free conditions
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from frozen colonic tissues was extracted using Qiagen RNeasy Universal kit. Integrity of the isolated RNA was analysed using the Agilent TS HS RNA Kit and TapeStation 4200
1000ng of total RNA was treated with the NEBNext poly (A) mRNA Magnetic Isolation Module (NEB, #E7490L). RNA sequencing libraries were produced by using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770L). Quantification of the library was performed using a dsDNA HS Assay Kit and Qubit (Molecular Probes, Life Technologies) and qualification was done using the Agilent TS D1000 kit and TapeStation 4200. 250nM of each library was pooled together and diluted to 4nM according to the NextSeq manufacturer’s instructions. 1.6pM was loaded onto the Flow Cell with 1% PhiX library control. Libraries were sequenced with the Illumina NextSeq 550 platform with single-end reads of 75 cycles according to the manufacturer's instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
S6
|
| Data processing |
The RNA sequencing data were analyzed using the RASflow pipeline mapping the reads to the transcriptome (Mus_musculus.GRCm39.cdna) using the hisat2 aligner and counting features using featureCounts. For the genome and transcriptome datasets, any missing gene names (using mmusculus_gene_ensembl) are replaced with gene IDs and unnecessary columns like 'gene_id' are dropped. We filtered out sparse genes, which we defined as those with over 50% zero values, to concentrate on genes with meaningful expression levels and to decrease computational complexity. We started with 35453 genes and removed 14981 sparse genes, leaving us with 20472 genes. Following this, we normalized the data with a scaling factor of 10^6. The purpose of this normalization was to make the samples comparable by compensating for variations in library sizes.
Assembly: Mus_musculus.GRCm39.cdna
Supplementary files format and content: Xlsx file includes RPKM values for each Sample
|
| |
|
| Submission date |
Feb 29, 2024 |
| Last update date |
Mar 19, 2024 |
| Contact name |
Shai Bel |
| E-mail(s) |
shai.bel@biu.ac.il
|
| Organization name |
Bar-Ilan University
|
| Department |
The Azrieli Faculty of Medicine
|
| Street address |
8 Henrietta Szold St, P.O. Box 1589
|
| City |
Safed |
| ZIP/Postal code |
1311502 |
| Country |
Israel |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE260592 |
Antibiotics damage the colonic mucus barrier in a microbiota-independent manner I |
| GSE270467 |
Antibiotics damage the colonic mucus barrier in a microbiota-independent manner |
|
| Relations |
| BioSample |
SAMN40204015 |
| SRA |
SRX23855541 |
