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| Status |
Public on Jun 13, 2023 |
| Title |
NP137-treated LKPR sample 1 |
| Sample type |
SRA |
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| Source name |
skin
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| Organism |
Mus musculus |
| Characteristics |
tissue: skin
genotype: Lgr5CreER/ KrasLSL-G12D/p53fl/fl/Rosa26-YFP+/+
treatment: NP137 In vivo IP
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| Treatment protocol |
To determine the effect of anti-Netrin-1 molecule (NP137) on primary tumor, LKPR mice were treated intraperitoneally at 10 mg/kg every two days from 4 weeks after Tamoxifen injection and until the death of animal.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Control and NP137-treated skin tumors from LKPR mice were dissected, rinsed and digested in collagenase type I (Sigma) at 3,5mg/ml in HBSS (Gibco) for 1h at 37°C on a rocking plate protected from the light. Collagenase was blocked by the addition of EDTA (5mM) and then the cells were rinsed 1 time in PBS supplemented with 10% FBS and the cell suspension were filtered through a 70-μm cell trainer (BD Bioscience). For the wash next and antibody incubation, cells were resuspended in PBS supplemented with 2% FBS (facs buffer). Cells were incubated with BV711- conjugated anti-EPCAM (rat, clone G8.8, BD Bioscience Cat#563134, dilution 1:100), PE-conjugated anti-CD45 (rat, clone 30-F11, BD, Cat#553081, dilution 1:100) and PE-conjugated anti-CD31 (rat, clone MEC 13.3, BD, Cat#553373, dilution 1:100) antibodies for 30 min at 4 °C protected from light. For cell sorting, cells were also filtered through a 40-μm cell trainer (BD Bioscience). Living single tumor cells were selected by forward and side scatter, doublet discrimination and Hoestch exclusion. Profile of the tumors was analyzed following viability and profile of YFP, Lin- (CD45/CD31) and EPCAM expression. Once screened all lived cells were sorted for single cell RNA sequencing experiment using FACSAria and FACSDiva software (BD Bioscience)
10,000 single cells from the LKPR tumor were loaded onto each channel of the Chromium Single Cell 3′ microfluidic chips (V2-chemistry, PN-120232, 10X Genomics) and barcoded with a 10X Chromium controller according to the manufacturer’s recommendations (10X Genomics). RNA from the barcoded cells was subsequently reverse transcribed, followed by amplification, shearing 5′ adaptor and sample index attachment. The libraries were prepared using the Chromium Single Cell 3′ Library Kit (V3-chemistry, PN-120233, 10X Genomics) and sequenced on an Illumina Novaseq 6000 (paired-end 100bp reads).
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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 |
10x Genomics
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| Data processing |
Quality control and downstream analysis were performed using the Seurat R package (v.3.1.5). For each sample, all of the cells passed the following criteria: showed expression of more than 600 and less than 8000 unique genes and had less than 25% UMI counts belonging to mitochondrial sequences.
Read counts were normalized by NormalizeData function of Seurat, with parameter ‘normalization.method = LogNormalize and scale.factor=10000’.
A PCA for each sample was calculated using the scaled expression data of the most variable genes (identified as outliers on a mean/variability plot, implemented in the FindVariableGenes).
UMAP calculation and graph-based clustering were done for each sample using the appropriate functions from Seurat (default parameters) with the respective PCA results as input.
In addition, to verify proliferating stages, the S-phase and GS/M-phase scores were regressed out by CellCycleScoring function, implemented in the Seurat.
Assembly: mm10-3.0.0
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Jun 06, 2023 |
| Last update date |
Jun 13, 2023 |
| Contact name |
Sebastiaan Vanuytven |
| E-mail(s) |
sebastiaan.vanuytven@ulb.be
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| Organization name |
Université Libre de Bruxelles
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| Street address |
Campus Erasme CP 637
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| City |
Brussels |
| ZIP/Postal code |
1000 |
| Country |
Belgium |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE234265 |
Pharmacological targeting Netrin-1 inhibits EMT in cancer [scRNA-seq] |
| GSE234267 |
Pharmacological targeting Netrin-1 inhibits EMT in cancer |
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| Relations |
| BioSample |
SAMN35648225 |
| SRA |
SRX20602724 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7461401_IPA1-Netrin-190913_barcodes.tsv.gz |
86.7 Kb |
(ftp)(http) |
TSV |
| GSM7461401_IPA1-Netrin-190913_features.tsv.gz |
272.8 Kb |
(ftp)(http) |
TSV |
| GSM7461401_IPA1-Netrin-190913_matrix.mtx.gz |
90.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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