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| Status |
Public on May 08, 2024 |
| Title |
control_FOXO1_non-stimulated_stimulated1 |
| Sample type |
SRA |
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| Source name |
T cell
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| Organism |
Homo sapiens |
| Characteristics |
cell type: T cell
donor: Healthy donor
genetic modification: α-Lewis-Y-CAR
genotype: control, FOXO1
treatment: no stimulation, stimulation with MCF7 tumour cells
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| Treatment protocol |
Cultured without stimulation (non-stimulated), stimulated with MCF7 tumour cells for 24 h
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| Growth protocol |
Human peripheral blood mononuclear cells (PBMCs) were isolated from normal buffy coats using Ficoll centrifugation and PBMCs were cultured in RPMI supplemented with 10% FCS, sodium pyruvate, glutamax, NEAA, HEPES and penicillin/streptomycin. PBMCs were activated with antihuman CD3 (30ng/ ml) and IL-2 (600 IU/ml) for 48 hours before transduction with a lentvirus encoding a second generation scFv–anti–Lewis-Y CAR (41BBz and CD3ζ). Cells were cultured at a density of 1x106/ml following the transduction process.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Protocol for capture: Chromium Next GEM single cell 5’reagent kit v2
Protocol for gene expression and VDJ Library prep: Chromium Next GEM single cell 5’reagent kit v2 ( including Fragmentation, Ligation, Indexing)
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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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| Data processing |
scRNA-seq data were generated using the 10x Cell Ranger pipeline (7.1.0) and hg38 genome. Specifically, cellranger multi was used to generate raw feature barcode matrices. Downstream analysis was performed in R (version 4.2.0). Empty droplets were detected and removed from the raw feature barcode matrix using the emptyDrops function from the DropletUtils (version 1.16.0) package and doublets were detected and removed using DoubletFinder (verison 2.0.3). Using Seurat (version 4.3.0), cells with less than 200 features and more than 5% mitochondrial reads were excluded. Standard Seurat data processing and normalization steps were performed: NormalizeData, FindVariableFeatures, ScaleData, RunPCA, RunUMAP, FindNeighbors and FindClusters; clusters with low-quality metrics were removed, and the final resolution was determined using results from the clustree package (version 0.5.0). LMOs were demultiplexed using HTODemux (Seurat). DEGs were calculated using the functions FindAllMarkers (Seurat) using a log2 fold change threshold of 0.125 and an adjusted P value of less than 0.05, and included the number of counts as a latent variable. Pseudobulk DEGs were detected using the Libra package (version 1.0.0) using the run_de function. Geneset enrichment was performed using the fgsea package with all expressed genes as the background gene list, which was ranked by average log fold change detected with FindMarkers using a log2 fold change threshold of 0 and min.pct parameter set to 0. To perform diffexp analyses and GSEA between individual groups within each cluster, the to_psuedobulk function from Libra was used to pull out pseudobulk count matrix of each replicate pool and clusters. EdgeR and fgsea was then utilized to perform differential expression and gsea analyses of reference gene signatures. The single-cell signature explorer program was utilized for visualization of gene signatures across UMAP plots.
Assembly: hg38 genome
Supplementary files format and content: Tab-separated values files and matrix files (output from Cellranger)
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| Submission date |
Apr 04, 2024 |
| Last update date |
May 08, 2024 |
| Contact name |
Kevin C sek |
| E-mail(s) |
[email protected]
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| Phone |
0432960331
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| Organization name |
Peter MacCallum Cancer Centre
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| Department |
Cancer Immunology
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| Lab |
Beavis Lab
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| Street address |
305 Grattan Street
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| City |
Melbourne |
| State/province |
VIC |
| ZIP/Postal code |
3000 |
| Country |
Australia |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE225527 |
Overexpression of FOXO1 enhances CAR T cell polyfunctionality, metabolic fitness and efficacy against solid tumors |
| GSE263256 |
FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy [human_FOXO1_scRNAseq] |
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| Relations |
| BioSample |
SAMN40753153 |
| SRA |
SRX24159873 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8188693_Pooled1_barcodes.tsv.gz |
2.4 Mb |
(ftp)(http) |
TSV |
| GSM8188693_Pooled1_features.tsv.gz |
287.7 Kb |
(ftp)(http) |
TSV |
| GSM8188693_Pooled1_matrix.mtx.gz |
263.6 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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