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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 25, 2018 |
| Title |
mLN-SPF-1 |
| Sample type |
SRA |
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| Source name |
mesenteric lymph node
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| Organism |
Mus musculus |
| Characteristics |
cell type: fibroblastic reticular cell
tissue: mesenteries
organ: lymph node
housing: specific-pathogen-free
strain: Balb/c
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| Treatment protocol |
For stromal cell isolation, skin-draining pLNs (inguinal and axillary) or mLNs were resected and digested in RPMI 1640 medium (Gibco) containing 0.2 mg/ml collagenase P (Roche), 0.15 U/ml dispase (Roche) and 0.2 mg/ml DNase I (Roche). After digestion, cells were kept at 4°C in PBS containing 0.2 % BSA and 5 mM EDTA (Roth). In a first step, CD45- cells were enriched by autoMACS separation after indirect magnetic labeling of CD45+ cells using anti‑CD45-APC (30-F11, eBioscience) followed by anti‑APC microbeads (Miltenyi Biotec). Subsequently, the CD45- fraction was stained using fluorescence-coupled antibodies and either analyzed directly by flow cytometry or used to sort CD45-Ter119-CD31-gp38+ FRCs by FACS (Aria II, 100 μm nozzle; purity >91‑97 %) for RNA‑seq analysis.
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| Growth protocol |
ex vivo
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from ex vivo sorted FRCs via DNA/RNA AllPrep Kit (Qiagen) according to the manufacturer's protocols.
Quality and integrity of total RNA was controlled on Agilent Technologies 2100 Bioanalyzer. Purification of poly A-containing mRNA was carried out via poly T oligo attached magnetic beads. Following purification, the mRNA was subjected to library preparation using Script Seq v2 Library preparation kit (Epicentre).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Libraries were aligned versus the mouse reference genome (assembly: GRCm38) using the splice junction mapper Tophat2 v1.2.0 with default parameterization.
Reads aligned to annotated genes were quantified with the htseq-count program [Anders, Pyl, Huber, 2015, Bioinformatics 31, 166-169].
Determined read counts served as input to DESeq2 [Love, Huber, Anders, 2014, Genome Biology 15, 550] for pairwise detection and quantification of differential gene expression.
RPKM (reads per kilobase maximal transcript length per million mapped reads) values were computed for each library from the raw gene counts.
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample.
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| Submission date |
Mar 21, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Joern Pezoldt |
| E-mail(s) |
jorn.pezoldt@epfl.ch
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| Phone |
0041766040171
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| Organization name |
EPFL
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| Department |
SV
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| Lab |
Laboratory Systems Biology and Genetics
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| Street address |
Station 19, SV 3818.A
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| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE79435 |
Neonatally imprinted mesenteric lymph node stromal cell subsets induce tolerogenic dendritic cells [endogenous FSCs] |
| GSE116633 |
Neonatally imprinted mesenteric lymph node stromal cell subsets induce tolerogenic dendritic cells |
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| Relations |
| BioSample |
SAMN04570634 |
| SRA |
SRX1650257 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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