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| Status |
Public on Mar 01, 2023 |
| Title |
NOS1 KO +LPS 4h, replicate 1 |
| Sample type |
SRA |
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| Source name |
lung
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| Organism |
Mus musculus |
| Characteristics |
cell type: hematopoietic (CD45+ enriched)
tissue: lung
strain: mouse
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| Treatment protocol |
WT or NOS1-/- mice were challenged with intraperitoneal dose of 10 mg/kg LPS or vehicle control for 4 h. CD45+ cells were isolated from lungs using EasySepTM Mouse CD45 Positive Selection Kit
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| Extracted molecule |
total RNA |
| Extraction protocol |
whole lungs were minced and digested with 500 μg/mL Liberase TB (Sigma, # 5401127001), 150 μg/mL DNase I (Sigma #1010459001), and 5 mM MgCl2 in PBS (pH 7.4) for 20 min at 37 oC. Then, single cell suspension was filtered through 70 μm nylon mesh cell strainer and treated with RBC Lysis Buffer (Invitrogen, # 00430054) according to manufacturer’s instructions. Ten million lung cells in suspension were mixed with 5 μL Component A, 5 μL Component B, and 5 μL magnetic RapidSpheresTM provided in the EasySep kit, and incubated for 5 min in ice. CD45+ cells trapped in magnetic beads were isolated using a magnetic platform, and released diluted in EasySepTM Buffer (StemCell #20144).
Sample quality, library complexity, and alignment statistics were checked using an established pipeline at the NUSeq Core Facility at Northwestern University
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
10x Genomics
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| Data processing |
Demultiplexed raw reads were mapped to the mouse genome (Mm10) using Cell Ranger (v6.1.0) with default settings.
Unfiltered count matrices for each library were loaded into R for processing by the Seurat package (V4.1.0, Hao and How et al. 2021) within R (v4.1.3) (Hao et al., 2021). Several filters were applied using Seurat. First, cells were removed if they didn’t have a minimum of 300 features. Then we applied filters to remove cells that had more than 20% mitochondrial genes, or had fewer than 700 features, or had either fewer than 1000 UMIs, or had more than 100,000 UMIs. Applying these filters reduced the number of combined cells from 64,330 to 58,011. Cell cycle stage was estimated using Seurat. All samples were combined into a single Seurat object and then log normalized. Cell cycle stage, percentage of mitochondrial genes, and number of features were regressed out during normalization.
Dimensionality reduction for clustering was done via PCA combined with Jackstraw in the Seurat package. Clustering was performed using the first 50 PCs with a range of resolutions (0.25 – 4, stepping every 0.5) and then used as input to t-SNE for imaging. The t-SNE plots were consulted to select a clustering resolution of 0.75. The Seurat function FindAllMarkers() was used to identify marker genes that differentiate any given cluster from the rest of the cells. Another Seurat function, FindMarkers(), was used to compare individual clusters to each other and to find genes that differentiate the two clusters
SingleR (v1.6.1) was used to identify resident macrophage cells in the dataset (Aran et al., 2019). We based the cell-type classification on the ImmGen database, which has normalized expression values for immune cells from 830 murine microarrays. Macrophage populations were confirmed with UMAP plotting of canonical marker genes
String network was generated using the list of differentially expressed genes in macrophage subclusters (iAM vs rAM), uploaded to Cytoscape 3 against reference species (Mus musculus) with confidence cutoff of 0.4 and zero additional interactions. Gene networks were clustered using MCL algorithm with granularity of 4. String functional enrichment was retrieved for network using the whole genome as background.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Feb 22, 2023 |
| Last update date |
Mar 03, 2023 |
| Contact name |
Brian Wray |
| E-mail(s) |
brianwray26@gmail.com
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| Organization name |
Northwestern University
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| Department |
Feinberg School of Medicine
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| Street address |
633 Clark Street
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| City |
Evanston |
| State/province |
IL |
| ZIP/Postal code |
60208 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE225834 |
SOCS1 regulates a subset of NFκB-target genes through direct chromatin binding and defines macrophage functional phenotypes (scRNA-Seq). |
| GSE225835 |
SOCS1 regulates a subset of NFκB-target genes through direct chromatin binding and defines macrophage functional phenotypes |
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| Relations |
| BioSample |
SAMN33417448 |
| SRA |
SRX19474201 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7057444_KO_LPS1_barcodes.tsv.gz |
59.3 Kb |
(ftp)(http) |
TSV |
| GSM7057444_KO_LPS1_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
| GSM7057444_KO_LPS1_matrix.mtx.gz |
91.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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