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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 22, 2022 |
| Title |
Human_scRNA-seq_K297_APC |
| Sample type |
SRA |
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| Source name |
Tumor
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Tumor
cell type: Antigen presenting cells
disease state: ccRCC
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell sorting was performed byFACS Aria II (BD Biosciences). For scRNA-seq (10x) of mouse samples live, CD3-CD19-Ly6G+ I-A/I-E+ cells were sorted from naïve LN, tumor-draining LN, and TRAMPC1-GP tumors. For human scRNA-seq (10x) fresh kidney tumors were used, antigen-presenting cells were sorted as live, CD45+, CD3-, HLADR+
Single cell RNAseq libraries were generated using the Chromium Single Cell 5’1007Library & Gel Bead Kit (10x Genomics) according to the manufacturer’s protocol. Briefly, cells were sorted and captured into the Gel Beads-in-emulsion (GEMs). After reverse transcription, the GEMs were disrupted and the barcoded cDNA was isolated, pooled, and amplified by PCR (13cycles). The amplified cDNA was fragmented, and subjected to end repair and A-tailing followed by a sample index PCR (16 cycles). The purified libraries were sequenced to a depth of 50,000 reads/cell on a 1013HiSeq3000 (Illumina) with 26 cycles for read 1, 8 cycles for index 1 (i7), and 91 cycles for read 2.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
Cell sorting was performed byFACS Aria II (BD Biosciences). For scRNA-seq (10x) of mouse samples live, CD3-CD19-Ly6G+ I-A/I-E+ cells were sorted from naïve LN, tumor-draining LN, and TRAMPC1-GP tumors. For human scRNA-seq (10x) fresh kidney tumors were used, antigen-presenting cells were sorted as live, CD45+, CD3-, HLADR+
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| Data processing |
Alignment, filtering, barcode counting, and unique molecular identifier counting were performed using Cell Ranger v3.1. Data were further analyzed using Seurat v.3.074. Briefly, cells with a percentage of mitochondrial genes below 0.05% were included. Cells with more than 4,000 or less than 1,000 detected genes were considered as outliers and excluded from the downstream analyses. Raw unique molecular identifier counts were normalized to unique molecular identifier count per million total counts and log-transformed. Variable genes were selected based on average expression and dispersion. PCA was performed using variable genes. Clusters were identified using the shared nearest neighbor algorithmin Seurat and t-SNE plots were generated based on selected principal component analysis dimensions. Marker genes were identified by the Seurat function FindAllMarkers. Log normalized data are shown in the form of feature plots with the scale in a range of0 (grey) to 2.5~5 (purple). Gene set scoring was performed using the VISION R package v.1.1.0. Briefly, expression of signature genes is weighted based on predicted dropout probability calculated from nearest neighbors, and the normalized expression summed for all genes in the gene set.
Assembly: HG38
Supplementary files format and content: Gene name and raw counts for sequenced cells for each sample
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| Submission date |
Dec 20, 2022 |
| Last update date |
Jan 04, 2023 |
| Contact name |
Haydn Kissick |
| E-mail(s) |
haydn.kissick@emory.edu
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| Phone |
6172598364
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| Organization name |
Emory University
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| Street address |
1462 Clifton Rd
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| City |
Atlanta |
| State/province |
Georgia |
| ZIP/Postal code |
30322 |
| Country |
USA |
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| Platform ID |
GPL21290 |
| Series (1) |
| GSE216731 |
LN-stem, tumor stem, tumor terminally differentiated CD8 T cells from human kidney cancer |
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| Relations |
| BioSample |
SAMN32330244 |
| SRA |
SRX18916767 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6862354_K297_named.csv.gz |
5.3 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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