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| Status |
Public on Sep 01, 2023 |
| Title |
Met2_173 |
| Sample type |
SRA |
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| Source name |
Metastatic brain with MDA-MB-231BR2
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
mouse host genotype: Foxn1 nu/nu
mouse diagnosis: Breast cancer brain metastasis
tissue: Brain
cell type: Astrocytes (CD45-, ACSA2+), myeloid cells (CD45+ CD11b+), and 231BR cells
batch: Group 3
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single cell suspension from mouse brain was prepared using the Adult Brain Dissociation Kit, Mouse and Rat (Miltenyi Biotec) with some modifications. Briefly, whole dissected brains were chopped into 8 pieces of equal size and placed into C tube (Miltenyi Biotec) containing enzyme P and A. Brain tissue was digested using gentleMACS Octo Dissociator with heaters operating the Adult brain dissociation protocol (Miltenyi Biotec). After digestion, the cell suspension was strained over a sterile 70μm strainer (Fisher Scientific) and washed with 5mL FACS buffer containing ice cold DMEM/F12, 50mM HEPES, and 2% BSA. After removal of myelin by density centrifugation, the cell pellet was washed and remaining red blood cells were lysed with red blood cell lysis buffer. Cells were then re-suspended in FACS buffer and blocked with anti-CD16/32 for 15 minutes on ice. Next, cells were stained with fluorescent antibodies on ice for 15 minutes shielded from light. The labeled cells were washed with 500μL of FACS buffer and resuspended in 500μL of FACS buffer, strained through 40μm strainer prior to sorting on BD FACSAria Fusion sorter. For sorting of microglia, astrocytes, and cancer cells, cells were gated for size based on forward and side scatter, single cells, and Sytox Blue viability (Thermofisher, S34857). All myeloid cells (CD45+ CD11b+) and astrocytes (CD45-, ACSA2+) were sorted from control and metastatic mouse brains into 500μL of chilled FACS buffer. GFP+ 231BR cells were sorted from metastatic brains into 500μL of FACS buffer.
FACS isolated mouse microglia cells were centrifuged for 10 minutes at 300g and washed with 0.04% BSA in PBS. Cells were resuspended to achieve approximately 1,000 cells/µL. Final cell suspensions were counted on the Countess II automated cell counter to determine actual concentration for droplet generation. Cells were loaded onto the 10x Genomics Chromium Single Cell Gene Expression 3’ v2 Chemistry kits for GEMs generation. Following the Chromium Single Cell 3′ Reagents Kits version 2 user guide (CG00052 Rev B), cells were loaded to achieve approximately 10,000 cells for capture. Libraries were sequenced on the Illumina HiSeq 4000 platform to achieve an average of read depth of 50,000 mean reads per cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Single-cell RNA-Seq [10X Genomics]
PolyA RNA
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| Data processing |
Sequencing reads were aligned utilizing 10x Genomics Cell Ranger Count 3.0.2 to the dual indexed mm10/GRCh38 genome provided by Cell Ranger.
Cells in dataset were subsequently trimmed and analyzed in Seurat v3, with assigned cell type labels and quality control metrics for each cell passing initial quality filtering included in the metadata sheets.
Genome_build: mm10/GRCh38
Supplementary_files_format_and_content: Space-delimited text files contain a matrix of counts; rows are genes and columns are samples.
Supplementary_files_format_and_content: TSV/MTX sets are output from the Cell Ranger pipeline and can be used to generate a matrix using Seurat's Read10X function.
Supplementary_files_format_and_content: Metadata sheets include quality control and cell type label information for the cells analyzed in the associated study.
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| Submission date |
Apr 01, 2020 |
| Last update date |
Sep 01, 2023 |
| Contact name |
Devon Lawson |
| Organization name |
University of California, Irvine
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| Street address |
839 Health Science Road, Sprague Hall 140
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| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92612 |
| Country |
USA |
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| Platform ID |
GPL25431 |
| Series (2) |
| GSE147942 |
Microglia coordinate the anti-tumor immune response to breast cancer brain metastasis [Foxn1 nu/nu 10X data] |
| GSE147949 |
Microglia coordinate the anti-tumor immune response to breast cancer brain metastasis |
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| Relations |
| BioSample |
SAMN14521673 |
| SRA |
SRX8044354 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4450697_Met2_173_barcodes.tsv.gz |
43.4 Kb |
(ftp)(http) |
TSV |
| GSM4450697_Met2_173_features.tsv.gz |
626.1 Kb |
(ftp)(http) |
TSV |
| GSM4450697_Met2_173_matrix.mtx.gz |
84.8 Mb |
(ftp)(http) |
MTX |
| GSM4450697_Met2_173_metadata.txt.gz |
162.6 Kb |
(ftp)(http) |
TXT |
| GSM4450697_Met2_Foxn1_mm10grch38_untrimmed_matrix.txt.gz |
32.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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