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| Status |
Public on Feb 26, 2021 |
| Title |
tumor - HSF1-303-7 |
| Sample type |
SRA |
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| Source name |
tumor
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| Organism |
Mus musculus |
| Characteristics |
genotype: HSF1-S303/7A
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tumors from HSF1S303/7A and WT mice were dissected, manually dissociated, and digested enzymatically with 0.04 U/mL Liberase TM (Sigma) and 0.15 mg/ml Dnase I (Roche) in Hank’s Balanced Salt Solution (Life Technologies) for 30 min at 37 °C with agitation. Tumors were homogenized by briefly vortexing and filtered through a 70-μm nylon filter. Single-cell suspensions were washed with 1 × PBS-4% FBS before incubation 20 min on ice at 5 × 107 cells/ml with 500 ng/ml Fc block (2.4G2, BD Pharmingen) to prevent nonspecific antibody binding. Cells were then incubated 1 h on ice with PE-eFluor610-conjugated CD45 monoclonal antibody (30-F11, eBioscience). For scRNAseq libraries, DAPI- (live) CD45+ and CD45− cells were sorted using a SY3200 flow cytometer. Sorted cells were counted, and live CD45+ and CD45− cells were mixed 5:1.
The libraries were prepared using the Chromium Single Cell 3′ Reagent Kits (v2, 10x Genomics): Single Cell 3′ Library & Gel Bead Kit v2 (PN-120237), Single Cell 3′ Chip Kit v2 (PN-120236) and i7 Multiplex Kit (PN-120262) and following the Single Cell 3′ Reagent Kits (v2) User Guide (manual part no. CG00052 Rev A).
single-cell RNA-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The Cell Ranger Single-Cell Software Suite (v3.0.0) (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) was used to perform sample demultiplexing, barcode processing and single-cell 3′ gene counting
The cDNA insert was aligned to the mm10/GRCm38 reference genome. Only confidently mapped, non-PCR duplicates with valid barcodes and unique molecular identifiers were used to generate the gene-barcode matrix that contained 7847 and 5289 cells for HSF1-WT and HSF1-S303/7A, respectively.
Further analysis—including quality filtering, the identification of highly variable genes, dimensionality reduction, standard unsupervised clustering algorithms and the discovery of differentially expressed genes—was performed using the Seurat R package v3.0.
To exclude low-quality cells, we removed 2% cells that expressed the lowest number of genes. To exclude cells that were extreme outliers in terms of library complexity and that might possibly include multiple cells or doublets, we first calculated the distribution of genes detected per cell and removed any cells in the top 2% quantile and then calculated a doublet score per cell using doubletCells from the scran Bioconductor package (v1.12.0).
After quality filtering, 6654 and 4802 cells for HSF1WT and HSF1S303/7A, respectively, remained. After removing unwanted cells from the dataset, we normalized cell expression by the library size, multiplied by a scale factor of 10,000 and log-transformed the result.
Genome_build: mm10
Supplementary_files_format_and_content: counts-HSF1-303-7.tar.gz and counts-HSF1-WT.tar.gz are the counts for each sample produced by Cell Ranger. The counts.normalized.csv.gz consists of integrated and normalized counts after all quality filtering steps. The metadata.csv consists of the metadata, including clusters and cell types.
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| Submission date |
Feb 25, 2021 |
| Last update date |
Feb 26, 2021 |
| Contact name |
Kathryn Grace Hockemeyer |
| Organization name |
NYU Langone Health
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| Department |
Pathology
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| Lab |
Aifantis
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| Street address |
550 1st Avenue
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10016 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE167551 |
The NK cell stress response status modulates anti-tumor immunity [scRNA-seq] |
| GSE167552 |
The stress response regulator HSF1 modulates natural killer cell anti-tumour immunity |
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| Relations |
| BioSample |
SAMN18058570 |
| SRA |
SRX10170265 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5107421_counts-HSF1-303-7.tar.gz |
57.5 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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