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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 09, 2023 |
| Title |
Mouse #66691 scRNA-seq |
| Sample type |
SRA |
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| Source name |
Melanoma single cell suspension
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| Organism |
Mus musculus |
| Characteristics |
genotype: WT
Sex: F
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| Extracted molecule |
total RNA |
| Extraction protocol |
Resected BRAFV600E/PTEN-deficient melanomas were cut into 1-2mm fragments and digested using the Tumor Dissociation Kit, mouse (Miltenyi) in DMEM using the soft/medium protocol according to manufacturer’s instructions. RBC lysis was performed in ACK buffer for 5 minutes on ice, followed by a wash in 1X DPBS containing 0.04% BSA. We applied droplet-based scRNA-seq (Chromium, 10X Genomics) to single-cell suspensions of BRAFV600E/PTEN-deficient melanomas from 3 WT and 3 dKO mice. 6 × 10^3 cells were loaded per well for each of the 6 samples, partitioning and library preparation were performed according to manufacturer’s protocol for the Chromium 3’ Gene Expression v3 kit.
Cells were processed according to Chromium 3’ Gene Expression V3 Kit (10X Genomics) using manufacturer’s guidelines followed by sequencing on a S1 NovaSeq chip (Illumina Inc.). Qubit 3 (Fisher Scientific) and 2100 Bioanalyzer (Agilent) were used for quality check of cDNA.
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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 |
66691_melanoma
scRNA-seq of melanoma single cell suspension at 35 days post induction
clustered_seurat_object.RData
annotated_seurat_object.RData
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| Data processing |
The output BAM file from sequencing was processed through cellranger software v7.0.1 (10X Genomics). Filtered gene-barcode matrices generated using the mm10-2020-A transcriptome (10X Genomics) were used for downstream analysis using Seurat 4.0.
Low-quality cells (less than 500 UMIs or 250 detected genes, or at least 20% mitochondrial transcripts) were removed
For each sample, data was normalized with SCTransform, regressing out cell cycle scores and mitochondrial ratio
Datasets were integrated using reciprocal PCA on the first 50 principal components with an alignment strength of 10.
Clusters were determined using a variety of resolutions and annotated using top cluster-specific genes conserved between WT and dKO cells, as well as known lineage genes for expected cell types. At final resolution (0.6), 3 clusters with a low number of significant genes, also displaying a low nuclear transcriptome complexity, were considered to represent low quality cells and were discarded. The least abundant cluster, characterized by genes of multiple lineages, likely contained doublets and was also discarded.
Assembly: mm10-2020-A transcriptome
Supplementary files format and content: Two tab-delimited text and one matrix file including raw counts values of each gene for each sample, used as input for Seurat. One RData Seurat object containing the integrated cells of all 6 samples, final clustering up to to removal of clusters representing low-quality cells. One RData Seurat object containing the annotated clusters retained for further analysis.
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| Submission date |
Apr 12, 2023 |
| Last update date |
Jun 09, 2023 |
| Contact name |
Dan Filipescu |
| E-mail(s) |
dan.filipescu@mssm.edu
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| Phone |
2128249265
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| Organization name |
Icahn School of Medicine at Mount Sinai
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| Department |
Oncological Sciences
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| Lab |
Emily Bernstein
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| Street address |
1470 Madison Ave
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE200751 |
MacroH2A restricts melanoma progression via inhibition of chemokine expression in cancer-associated fibroblasts. |
| GSE229530 |
MacroH2A restricts melanoma progression via inhibition of chemokine expression in cancer-associated fibroblasts [scRNA-seq] |
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| Relations |
| SRA |
SRX19947951 |
| BioSample |
SAMN34159610 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7164978_66691_melanoma_barcodes.tsv.gz |
33.7 Kb |
(ftp)(http) |
TSV |
| GSM7164978_66691_melanoma_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
| GSM7164978_66691_melanoma_matrix.mtx.gz |
63.3 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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