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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 20, 2024 |
| Title |
BLM2 |
| Sample type |
SRA |
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| Source name |
colon
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| Organism |
Mus musculus |
| Characteristics |
disease: colon tumor
group: BLM2
tissue: colon
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| Extracted molecule |
total RNA |
| Extraction protocol |
To obtain single cell solutions of tumor cells, pooled distal (Min, MSH2KO), distal and proximal (BLM) or proximal (BLM2) tumors were washed in HBSS, incubated in 0.25% trypsin-EDTA for 10 minutes at 37oC, followed by the addition of FBS to inactivate the trypsin. Next, the tumors were digested by incubation in liberase TM (0.05 mg/ml; Roche, #05401119001)+DNAse (0.2 mg/ml) in DMEM at 37oC for 2H while rotating. Following washing in HBSS and an additional incubation in 0.25% trypsin-EDTA for 10 minutes at 37oC, cells were resuspended in DMEM+10%FBS and filtered through a 40 mM mesh cell strainer. Cells were then washed with DPBS+0.1% BSA and viable cells were counted. Cells were resuspended at 1,000 cell per ml in DPBS+0.1% BSA
All single cell preparations used for sequencing had a viability of > 80%. 10,000 cells per sample were targeted for input to the 10X Genomics Chromium system using the Chromium Next GEM Single Cell 30 Kit v3.1 at the Indiana University School of Medicine (IUSM) Center for Medical Genomics core
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
Read alignment and gene-expression quantification of mouse scRNA-seq data was performed using the CellRanger Count pipeline (version 6.1.2, 10X Genomics)
Cells were filtered to include only cells with no more than 20% mitochondrial gene expression and doublets were removed using DoubletFinder. The data were normalized, and highly variable genes were identified and scaled using SCTransform. Next, dimensionality reduction by principal components (PCs) was calculated using RunPCA and to estimate the significant number of PCs to be used ElbowPlot function was used. Next, the uniform manifold approximation and projection (UMAP) embedding were calculated and visualized using RunUMAP and DimPlot. Unsupervised Louvain clustering was carried out using FindNeighbors and FindClusters.
In our analysis (Seurat_colon_1.RDS), we used Seurat v4.3.0.1 to perform batch-effect correction. 3000 highly variable genes were defined within the 4 mouse samples with the Seurat FindVariableFeatures function. We also identified unsupervised integration “anchors” for similar cell states using shared nearest neighbor graphs (FindIntegrationAnchors), and then integrated our 4 different datasets using these anchors using IntegrateData. The output was then transformed into principal component analysis (PCA) space for further evaluation and visualization
Assembly: mm10
Supplementary files format and content: RDS file
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| Submission date |
Dec 07, 2023 |
| Last update date |
Jun 20, 2024 |
| Contact name |
Ahmed H Ghobashi |
| E-mail(s) |
aghobash@iu.edu
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| Organization name |
Indiana University
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| Lab |
Heather O'Hagan
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| Street address |
1001 E. 3rd St
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| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47403 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE249625 |
Single-cell profiling reveals the impact of genetic alterations on the differentiation of inflammation-induced colon tumors |
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| Relations |
| BioSample |
SAMN38717126 |
| SRA |
SRX22818341 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7951958_BLM2_barcodes.tsv.gz |
62.2 Kb |
(ftp)(http) |
TSV |
| GSM7951958_BLM2_features.tsv.gz |
254.1 Kb |
(ftp)(http) |
TSV |
| GSM7951958_BLM2_matrix.mtx.gz |
92.1 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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