|
| Status |
Public on Sep 05, 2023 |
| Title |
Lgr5_m_O_rep1 (total RNA-seq) |
| Sample type |
SRA |
| |
|
| Source name |
Lgr5 positive cells from small intestine (intestinal stem cells)
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/J
cell type: Lgr5-ki-e-GFP-creER
Sex: male
age: old (18-26 months old)
|
| Growth protocol |
Young (2 months old) and old (18-26 months old) male and female wild-type C57BL6/J mice were group housed and maintained in a specific pathogen-free animal facility in Fritz Lipmann Institute with 12 h of light/dark cycle and fed with a standard mouse chow.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For the Lgr5-eGFP ISCs isolation, the villi-free intestinal pieces (2 cm) were incubated in 5mM EDTA/PBS on ice for 45 min. The tissue was then shaken vigorously for 1 min, the crypt solution was filtered through a 100 µm cell strainer and centrifuged at 300xg for 5 min at 4°C. The crypts were dissociated with the mixture of 18 ml TrypLE Express, 2 ml of 10X DNase I Buffer (100 mM Tris HCl pH 7.5, 25 mM MgCl2, 5 mM CaCl2) and 1 ml DNase I (10 mg/ml) for 30 min at 37°C with brief vortexing every 10 min. The single-cell suspension was then passed through a 20 µm cell strainer and centrifuged at 450xg for 5 minutes at 4°C. The pellet was resuspended in 3 ml FSM (2% FBS, 2.5 mM EDTA) containing 10 µM Y27632 and DAPI (1:1000). The single cell suspension was applied to FACS LSRII (BD Biosciences) and the Lgr5-eGFPHi ISCs were sorted for downstream analysis.
Total ribo-depleted RNA-seq library preparation was performed as described previously (Neri et al., Nature 2017) . In brief, 50 to 500 ng of total RNA were depleted of ribosomal RNA using the Ribo-Zero™ Gold Kit H/M/R Kit (illumina) following manufacturer’s instructions. Ribo-depleted RNA was resuspended in 17 μl of EFP buffer (illumina), heated to 94°C for 8 min, and used as input for first strand synthesis, using the TruSeq™ RNA Library Preparation Kit v2 (illumina) following manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
transcriptional profiling (RNA sequencing)
|
| Data processing |
Fastq files quality check was performed using FastQC v0.11.5.
The fastq files were mapped to the mm9 genome using TopHat v2.1.0 with the following parameters --bowtie1 --no-coverage-search -a 5.
The number of reads covered by each gene is calculated by HTSeq-Count 0.11.2 with -s no -a 0 -t exon -m intersection-nonempty parameters.
Genome_build: mm9
Supplementary_files_format_and_content: Htseq-count text file
|
| |
|
| Submission date |
Apr 09, 2019 |
| Last update date |
Sep 05, 2023 |
| Contact name |
Seyed Mohammd Mahdi Rasa |
| E-mail(s) |
[email protected]
|
| Phone |
015258164616
|
| Organization name |
Universitätsklinikum Schleswig-Holstein
|
| Department |
Institut for Immunology
|
| Lab |
Immunology (Prof. Sheffold)
|
| Street address |
Michaelisstrasse 5
|
| City |
Kiel |
| State/province |
Schleswig-Holstein |
| ZIP/Postal code |
24105 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE129510 |
Transcriptional and epigenetic landscape of the mouse intestine during aging identifies key molecular drivers of aging -associated dysfunctions and diseases. [Total RNA-seq - Lgr5] |
| GSE129713 |
Transcriptional and epigenetic landscape of the mouse intestine during aging identifies key molecular drivers of aging -associated dysfunctions and diseases |
|
| Relations |
| BioSample |
SAMN11371526 |
| SRA |
SRX5655948 |
