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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 03, 2024 |
| Title |
tumor cell spheroids day 4 |
| Sample type |
SRA |
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| Source name |
MC38-GFP
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| Organism |
Mus musculus |
| Characteristics |
cell type: MC38-GFP
treatment: none
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| Growth protocol |
MC38-GFP cells were from L.Borsig and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Gibco), 1% non essential amino acids (NEAA, Gibco) and 1% sodium-pyruvate (Gibco). BM cells were isolated by flushing the femurs and tibias of 8- to 10-week-old C57BL/6J mice and passed through a 70-μm filter. The BM cells were plated at a density of 3 x 105 cells/ml on tissue culture-treated 60 mm UpCell dish in complete RPMI-1640 medium supplemented with 100 ng/ml recombinant mouse M-CSF (PeproTech). On day 3, half of the medium was replaced. Cultures were treated on day 3 with 300 μM heme-albumin or albumin (as vehicle/control). For inflammatory polarization, INFγ (10 ng/ml, PeproTech) or LPS (10 ng/ml, Sigma) was added on day 6 for 24 hours. The BM cells were harvested for analysis on day 7 from the temperature responsive cell culture plates after cooling to room temperature. Cells were washed in PBS and centrifuged (300 g, 10 min) before processing.5 x 104 MC38-GFP cells +/- BMDMs in 1-1 ratio were seeded in 800 μl tumor cell medium with M-CSF (100 ng/ml) in 24-well microwell Sphericalplate® 5D (Kugelmeiers). 800 μl fresh culture cell medium was added on day 2-3.We conducted multiplexed scRNA-seq experiments with tumor-only and mixed cell type spheroids collected on days 4, 8, and 10 after formation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Spheroids were dissociated in 2 ml digestion medium (RPMI medium (Gibco) + 25 µg/ml Liberase ™ (Roche) + 40 µg/ml DNase I (Roche; 2000 U/ml) and incubated for 30-45 min in a water bath at 37°C with gentle shaking every 5 min. 4 ml PBS + 0.04% BSA was added to stop the digestion.
Single-cell suspensions were counted and transferred to 2 ml safe lock tubes and directly used for GEM generation following 10X Genomics protocol (CG000388 Demonstrated Protocol Chromium Next GEM Single Cell 3ʹ v3.1 (Dual Index) with Feature Barcode technology for Cell Multiplexing Rev B) targeting a cell recovery rate of 10’000 cells.
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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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| Data processing |
Read Alignment using CellRanger (version 7.0.0)
Preprocessing, Dimensionality reduction, Clustering (separate processing of conditions), remove cluster identified as macrophages
Functional classification of tumor cell clusters by means of DGE and GSEA using the MSigDB_Hallmark_2020 database
Assembly: Mus_musculus/GENCODE/GRCm39/Annotation/Release_M26-2021-04-20
Supplementary files format and content: .h5: output of cellRangerCount (raw and filtered bc_matrix)
Supplementary files format and content: .h5ad: processed data for tumor only spheroids, tumor cells of mixed spheroids, and macrophages of mixed spheroids
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| Submission date |
Jul 10, 2023 |
| Last update date |
Jan 03, 2024 |
| Contact name |
Florence Vallelian |
| E-mail(s) |
florence.vallelian@usz.ch
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| Organization name |
University Hospital Zürich
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| Street address |
Rämistrasse 100
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| City |
Zurich |
| ZIP/Postal code |
8091 |
| Country |
Switzerland |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE236995 |
Heme-TAMs prevent cancer cell apoptosis and drive growth, invasiveness, and metastasis |
| GSE237612 |
Hemorrhage-induced NRF2 signaling in tumor-associated macrophages drives cancer growth, invasion, and immunotherapy resistance |
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| Relations |
| BioSample |
SAMN36398094 |
| SRA |
SRX20970815 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7592776_TC4_filtered_feature_bc_matrix.h5 |
75.9 Mb |
(ftp)(http) |
H5 |
| GSM7592776_TC4_raw_feature_bc_matrix.h5 |
124.7 Mb |
(ftp)(http) |
H5 |
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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