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| Status |
Public on Jul 04, 2024 |
| Title |
CD8 T cells, Day 8 Armstrong |
| Sample type |
SRA |
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| Source name |
Spleen
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| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen
cell type: CD8
strain: C57BL/6
treatment: LCMV-Armstrong
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
C57BL/6 mice (3 biological replicates) were infected with LCMV-Arm. At 8 dpi, mice were sacrificed, and single cell suspensions generated from spleens followed by filtration through a 70 mM filter. Spleen samples were split into two halves: the first half was subjected to CD45+ cell isolation (Miltenyi Biotec #130-052-301) and the second half used for CD8+ T cell isolation (negative selection kit, STEMCELL Technologies #19853).
Single cell libraries were prepared using the 10X Genomics 5’ v2 single-cell RNA-seq kit following manufacturer instructions (10X Genomics). Sequencing was performed on an Illumina NovaSeq 6000 S2 flow cell (Illumina Inc., San Diego CA, USA) using 125 cycles - 26 bp for read 1 containing the cell barcode (16bp) and UMI (10bp) and 91 bp for read 2, containing the biological read.
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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 |
Mapping and quantification of expression profiles was conducted using salmon (v1.8.0) and GRCm39 (GENCODE rel M29). Salmon used the –alevin parameter with library orientation to ISF. Transcript level counts were imported using the tximeta (v1.14.0) in R (v4.2) and creation of a SingleCellExperiment object.
Quality control (QC) and normalization was completed by using scran (v1.24.0) and scater (v1.24.0). Briefly, cells with greater than 25% mitochondrial reads were removed and low quality cells as assessed by perCellQCMetrics in the scater package were removed using default guidelines. Normalization was first conducted using logNormCounts from the scuttle package (v1.6.2). Further normalization was implemented using the scater package. Doublet removal was conducted using computeDoubletDensity, a function in the scDblFinder package (v. 1.10.0).
After normalization and filtering of low quality cells, the top 10% of highly variable genes (HVGs) were used to run a PCA analysis using denoisePCA, a scran function, setting ncomponents equal to 2. Clustering analysis was achieved by using principle components and using buildSNNGraph function in scater to identify and build shared nearest neighbors, where k = 10 in that function. Cluster_walktrap function in igraph (v.1.3.2) was used for identifying clusters.
Assembly: mm10
Supplementary files format and content: quants_mat.gz, Data that contains the compressed count matrix
Supplementary files format and content: clustername_Seurat_filtered_SCE_Mito_out_no_doublets.rds, this the the SCE with all of the post QC methods used for the paper.
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| Submission date |
May 14, 2024 |
| Last update date |
Jul 04, 2024 |
| Contact name |
Brandon Oswald |
| E-mail(s) |
Brandon.Oswald@vai.org
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| Phone |
6168343380
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| Organization name |
Van Andel Institute
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| Department |
Metabolism
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| Lab |
Russell Jones
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| Street address |
333 Bostwick Ave NE
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| City |
Grand Rapids |
| State/province |
MI |
| ZIP/Postal code |
49503 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE267377 |
ACLY and ACSS2 link nutrient-dependent chromatin accessibility to regulation of CD8 T cell effector responses |
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| Relations |
| BioSample |
SAMN41389151 |
| SRA |
SRX24542523 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8264914_clustername_Seurat_filtered_SCE_Mito_out_no_doublets.rds.gz |
94.0 Mb |
(ftp)(http) |
RDS |
SRA Run Selector |
| Raw data are available in SRA |
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