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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 17, 2024 |
| Title |
WT1, B6, colon immune cells, scRNAseq |
| Sample type |
SRA |
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| Source name |
Colon
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| Organism |
Mus musculus |
| Characteristics |
tissue: Colon
cell type: CD45+ cells
genotype: WT
treatment: none
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| Growth protocol |
Colons from B6 mice were collected, cut into small pieces, and washed in gut buffer (1X Hank’s Balanced Salt Solution (HBSS) containing 2% heat-inactivated fetal bovine serum (FBS) and 15 mM of HEPES. Epithelial cells were removed by shaking in gut buffer with 5 mM EDTA for 30 minutes at 37°C, with occasional vortexing. Finally, tissues were treated with collagenase IV (20 mg/ml) and DNase I (10 mg/mL) in RPMI-1640 supplemented with 5% FBS and 15 mM of HEPES for 20 minutes at 37°C and passed through a 70 μM cell strainer to collect lamina propria cells. Cells were stained for viability using Zombie Viability Dye V500 (1:400), and surface stained with anti-CD45 APC (1:400, clone 30F11, Biolegend). Stained cell suspensions were sorted on a BD FACSAriaIII Cell Sorter (BD Biosciences) to obtain viable CD45+ cells.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Freshly sorted cells were washed and resuspended in 0.04% BSA in PBS for loading on the 10x Chromium G chip aiming to capture with 8000 cells per sample. Two mouse colons were processed and capture separately.
Single cell capture and cDNA preparation was done according to the 10x Genomics Single Cell 3’ RNA (v3.1) protocol. Libraries were sequenced on the NovaSeq 6000 (Illumina).
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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 Single Cell 3’ RNA (v3.1)
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| Data processing |
Raw gene expression matrices were generated for each sample by the Cell Ranger, and the output was analyzed using the Seurat package.
Low quality reads were filtered based on three criteria: number of detected genes per cell, number of UMIs expressed per cell and mitochondrial content, using the following threshold parameters: nGene (between 200 and 7500), nUMI (between 500 and 75,000), and percentage of mitochondrial genes expressed (< 7.5%).
Doublets were then identified by finding cells expressing markers of two cell lineages simultaneously, as well as using the DoubletFinder package.
Gene expression matrices were normalized using the NormalizeData function and scaled using the ScaleData function, regressing out effects of cell cycle and percentage of expressed mitochondrial genes. Identification of highly variable features, linear dimension reduction by PCA transformation, UMAP dimensionality reduction, and cell clustering were performed using standard Seurat package workflows.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Dec 05, 2023 |
| Last update date |
Jan 17, 2024 |
| Contact name |
David Langlais |
| E-mail(s) |
david.langlais@mcgill.ca
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| Organization name |
McGill University
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| Department |
Human Genetics
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| Lab |
Inflammation Genomics Lab
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| Street address |
740 Ave Dr Penfield, rm 4203, McGill Genome Centre
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| City |
MONTREAL |
| State/province |
QC |
| ZIP/Postal code |
H3A0G1 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE249342 |
CCDC88B interacts with RASAL3 and ARHGEF2 to regulate dendritic cell function in neuroinflammation and colitis |
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| Relations |
| BioSample |
SAMN38658928 |
| SRA |
SRX22756944 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7937245_B6_colon_scRNA_WT1_barcodes.tsv.gz |
40.2 Kb |
(ftp)(http) |
TSV |
| GSM7937245_B6_colon_scRNA_WT1_features.tsv.gz |
245.2 Kb |
(ftp)(http) |
TSV |
| GSM7937245_B6_colon_scRNA_WT1_matrix.mtx.gz |
62.1 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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