|
| Status |
Public on Jan 03, 2024 |
| Title |
LPS-treated BMDM + MC38-GFP spheroids (24h) |
| Sample type |
SRA |
| |
|
| Source name |
MC38-GFP & BMDM
|
| Organism |
Mus musculus |
| Characteristics |
cell type: MC38-GFP & BMDM
treatment: LPS
|
| 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).
|
| 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 multiplexed and then used for GEM generation following the 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. GEM generation and library preparation were performed according to the 10X Genomics protocol (CG000388).
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
combined_spheroids_day1_processed.h5ad
macrophages_processed.h5ad
combined_raw_feature_bc_matrix.h5
|
| Data processing |
Demultiplexing and read alignment using CellRangerMulti (cellranger-6.1.2)
Preprocessing: Filtering, normalization and log(x+1)-transformation, dimensionality reduction and UMAP
Assembly: Mus_musculus/GENCODE/GRCm39/Annotation/Release_M26-2021-04-20
Supplementary files format and content: .h5 output of CellRangerMulti
Supplementary files format and content: .h5ad containing processed data of the combined experiments
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| |
|
| Submission date |
Jul 05, 2023 |
| Last update date |
Jan 03, 2024 |
| Contact name |
Florence Vallelian |
| E-mail(s) |
florence.vallelian@usz.ch
|
| Organization name |
University Hospital Zürich
|
| Street address |
Rämistrasse 100
|
| City |
Zurich |
| ZIP/Postal code |
8091 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE236576 |
Mixed cell-type spheroids endorse the generation and maintenance of heme-TAMs in an experimental tumor microenvironment |
| GSE237612 |
Hemorrhage-induced NRF2 signaling in tumor-associated macrophages drives cancer growth, invasion, and immunotherapy resistance |
|
| Relations |
| BioSample |
SAMN36307928 |
| SRA |
SRX20898306 |
