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| Status |
Public on Jan 10, 2024 |
| Title |
MEKi_rep1 |
| Sample type |
SRA |
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| Source name |
human NRAS-mutated melanoma PDX in mice
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| Organism |
Homo sapiens |
| Characteristics |
protocol: xenograft
treatment: MEK-inhibitor
tissue: human NRAS-mutated melanoma PDX in mice
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| Extracted molecule |
total RNA |
| Extraction protocol |
Slow-frozen PDX tissue samples were quickly thawed in the water bath set to 37°C degree, re-suspended in 10 mL of ice cold RPMI with 1% BSA and spun down at 300g for 5 min. Further tissue samples were cut into small pieces and enzymatically digested for 30 min at 37 °C on a shaker with 5000U Collagenase IV, 15 KU DNAse I, 2 mL Accutase dissolved in RPMI with 2mM CaCl2 and 0.5% BSA 5 mL RPMI mixture. After incubation, digested tissue was filtered through 100 μm nylon and then through 35-μm cell strainers. For samples with viability below 80%, apoptotic and dead cells were removed using immunomagnetic cell separation with the Annexin Dead Cell Removal Kit and EasySepTM Magnet. If the cell pellet appeared red, red blood cell lysis was performed following the manufacturer's instructions. Cell number and viability was assessed on Luna-FLTMDual Fluorescence Cell counter using Photon slides by acridine orange propidium iodide stain and optimal cell concentrations were set to 700-1100 cells/μL according to 10x Genomics protocols.
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). 6000 cells per sample were targeted and libraries were diluted to 10 nM and pooled at balanced ratios according to the target cell number. Paired-end sequencing was performed on the Illumina NovaSeq S2 flow cell. According to 10x Genomics recommendation 50,000 read pairs per cell for GEX coverage was targeted.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
10x Genomics
counts.csv.gz
metadata.csv.gz
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| Data processing |
Demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software 3.1.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger). Raw count matrix has been generated using Seurat v4.1.1 after merging all samples. The cell-type calls in the metadata are output by SingleR v1.8.
Assembly: hg38
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Jan 05, 2023 |
| Last update date |
Jan 10, 2024 |
| Contact name |
Zsolt Balázs |
| E-mail(s) |
dzsidipi@gmail.com
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| Organization name |
University of Zurich
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| Street address |
Schmelzbergstrasse 26
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| City |
Zurich |
| ZIP/Postal code |
8006 |
| Country |
Switzerland |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE222258 |
Metabolic targeting as a strategy to overcome targeted therapy resistance in NRAS-mutated melanoma |
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| Relations |
| BioSample |
SAMN32604613 |
| SRA |
SRX18948047 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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