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| Status |
Public on Nov 12, 2021 |
| Title |
Steady_1-2 |
| Sample type |
SRA |
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| Source name |
splenic stroma
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6Jrj
sample type: ex vivo isolated cells
treatment: uninfected_naive
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| Treatment protocol |
Five-months-old C57BL/6Jrj female mice were i.p. injected with 10exp6 pfu of bacterial artificial chromosome–derived mCMV (pSM3fr-MCK-2 full-length; J Virol. 2011; 85(19):10346-53) or MOCK and sacrificed 24 hours p.i. Cells were sorted from pooled splenic preparations from 3 mice per condition.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single surface-decorated CD45-/+ITGB1hiCD31- cells were sorted by FACS ARIA II sorter (BD) and collected in PBS containing 0.04% w/v BSA at a densitiy of 400 cells/μl. Chromium™ Controller was used for partitioning single cells into nanoliter-scale Gel Bead-In-EMulsions (GEMs) and
Single Cell 3’ reagent kit v3 for reverse transcription, cDNA amplification and library construction (10xGenomics).
Sequencing was performed in paired-end mode (2 × 75 cycles) using NextSeq 500 sequencer (Illumina) to attain approximately 35,000 reads per single cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
RCM_steadyState_1.txt.txt.gz
SteadyState_1
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| Data processing |
Raw base call (BCL) files were demultiplexed to generate Fastq files using the cellranger mkfastq pipeline within Cell Ranger 3.0.3 (10x Genomics). Whole transcriptome Fastq files were processed using the standard cellranger pipeline within Cell Ranger 3.0.3 (10x genomics) against the merged genomes of GRCm38 (Ensembl v90) and MCMV (BAC derived wild type Smith strain, Accession Identifier: NC_004065). The expected cell parameter was set to 10,000 for all libraries. Briefly, cellranger count performs alignment, filtering, barcode counting, and UMI counting to obtain read count matrices (RCM). In total four RCMs were obtained, with two independent experiments performed for both virally infected and steady-state condition.
RCMs were further processed via R (version 3.6.2) using Seurat (version 3.1.5) and uwot (version 0.1.8). Cells with at least 1000 detected genes and < 7.5% mitochondrial reads were retained in the analysis. Analysis was performed in a stepwise manner to successively identify and focus on RPSC, WPSC and Pv/VSMC. Genes were included if they were expressed in at least 1 % of the cells.
Initially, the four RCMs were merged using FindIntegrationAnchors(list(experimental_batches), anchor.features = 1000, dims = 1:15, k.filter = 200, k.anchor = 10) and IntegrateData(). Data was scaled and regressed against the variables percent mitochondrial UMIs, percent ribosomal protein UMIs, percent viral UMIs and the total number of UMIs per cell. PCAs were computed using default settings. Uniform Manifold Approximation and Projection (UMAP) dimensional reduction via RunUMAP() and FindNeighbors() were performed using the first 15 PCA dimensions as input features. FindClusters() was computed at resolution 0.4. In total 50401 cells with 16426 detected genes were processed.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices for each replicate.
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| Submission date |
Aug 20, 2020 |
| Last update date |
Nov 12, 2021 |
| 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 |
GPL19057 |
| Series (1) |
| GSE156162 |
Single-cell transcriptional profiling of splenic fibroblasts reveals subset-specific innate immune signatures in homeostasis and during viral infection |
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| Relations |
| BioSample |
SAMN15867470 |
| SRA |
SRX8978680 |
| 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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