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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 08, 2024 |
| Title |
T1 Mammary_gland_tumour |
| Sample type |
SRA |
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| Source name |
Multiple_mammary_gland
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| Organism |
Mus musculus |
| Characteristics |
strain: FVB
genotype: MMTV-PyMT (Guy et al., 1992; Lin et al., 2003), Rosa-tdTomato (Madisen et al., 2010) and K14Cre (Dassule et al., 2000)
Sex: Female
age: 13-14-weeks-old
tissue: Mammary gland
condition: Tumour
animal_id: mouse 1
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| Growth protocol |
Mouse experiments were carried out on MMTV-PyMT (Guy et al., 1992; Lin et al., 2003), Rosa-tdTomato (Madisen et al., 2010) and K14Cre (Dassule et al., 2000), and back crossed in FVB background for 10 generations (99.9% FVB). Mice were fed ad libitum, handled and housed under the standard of the European Community Council Directive (2010/63/EU) and Spanish legislation. Mammary gland-specific expression of PyMT under the control of the MMTV promoter/enhancer in transgenic mice (MMTV-PyMT) results in widespread transformation of the mammary epithelium and in the development of multifocal mammary adenocarcinomas and metastatic lesions in the lymph nodes and in the lungs (Guy et al., 1992). Tumor formation and progression in these mice is characterized by four stages: hyperplasia, adenoma/mammary intra-epithelial neoplasia, and early and late carcinoma (Lin et al., 2003). K14-Cre transgene that has a human keratin 14 promoter directing expression of Cre recombinase, trigger deletion of flexed sequences in the endoderm and its derivatives (Dassule et al., 2000). Rosa-tdTomato is a universal Cre-responder transgene with strong, ubiquitous expression of tdTomato fluorescent protein (Madisen et al., 2010).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tumours where collected from Young adult (13-14-week-old) female MMTV-PyMT::K14Cre::Rosa-tdTomato mice, minced manually using sterile scalpels and further finely cut until homogeneous aspect using McIlwain Tissue Chopper (TED PELLA, INC). Minced samples where digested in digestion buffer (2,5ml for 1mg tumour) at 37ºC for 45 minutes on rocking plates chamber (reference VWR). Digestion buffer was based on PBS supplemented 2.5 Wünsch unit’s TH Liberase /ml (Roche) (50mg TH Liberase in 52 PBS = 5W unit/ml) and with 25μg/ml DNase I (Roche). To achieve single cells disruption, tumours fragments were pipetted up and down every 5 minutes. The progression of the cellular disaggregation was evaluate using microscope. Disaggregated samples were neutralized with 15ml breast medium supplemented by 25μg/ml DNase I and passed through a 70μm filter (BD Falcon) then 40μm filter (BD Falcon). Further, cells were spun down (5 minutes at 400 RCF at RT) and the pellet resuspended in 5ml of red blood lysis ACK (Ammonium-Chloride-Potassium) buffer for 5 minutes at RT with continuous gentINle rotation. After, cells were spun down and resuspended in 5ml FACS buffer/5mM EDTA and filtered through 40μm filter. 20μl of the cell preparation where mixed with equal volume of Trypan Blue and incubated for 5min to evaluate cells number and confirm the quality of single cells preparation, cell viability and low debris contains. Cells were further spun down (3 minutes at 400 RCF at RT) and resuspended in Dead cell removal beads (MiltenyiBiotec) at 100μl/10M and Incubated 15min at RT with gentle mixing. During the incubation, MACS LS column (MiltenyiBiotec) were placed on the magnet rack and conditioned by rinsing 1X with binding buffer 1X. After, cells were spun down (3 minutes at 400 RCF at 4C), resuspended in 3ml 1X binding buffer and applied on the magnetic column. Passing living cells (column retain the dead cells) were collected in 50ml Falcon tube placed in ice. Column where rinsed 4X with 3ml binding buffer to further living cells in the same Falcon tube. Collected cell were finally spun down (5 minutes at 400 RCF at 4C) and resuspended in cold breast medium and kept on ice. 20μl cells where mixed with 20ul trypan Bleu, incubate for 5minutes to count the proportion of viable and double check single cells state. Viability was first time evaluated on a BD FACS Aria III % as well as cancer cells, non-cancer cells and debris contains, and later, by measuring DAPI negative cells fraction. Finally, cells concentration was adjusted to 900-1000 cell/μl with cold FACS buffer and directly used for GEM (Gel Bead-In-Emulsions) preparation.
Individual cell encapsulation for single cell expression profiling was done using 10x Chromium Controller (10xGenomics). For every sample, single cell suspension was loaded into a Chromium Single Cell A Chip (10x Genomics) and processed following the manufacturer’s instructions. Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3’ Library & Gel Bead kit v2 and samples were multiplexed using Chromium i7 Multiplex kit (10x Genomics). Pooled libraries were then loaded on a HiSeq2500 instrument (Illumina) and sequenced to obtain 75 bp paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
single-cell gene expression profiling (scRNA-seq)
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| Data processing |
Sequencer: Illumina HiSeq 2500. HiSeq Sequencing v4 Chemistry. Read Length: 75 paired end.
Quality control of sequenced reads was performed using FastQC (Babraham Institute). Sequenced samples were processed using the Cell Ranger (v.2.2.0) pipeline (10x Genomics) and aligned to the CRGm38 (mm10) mouse reference genome customized to include reporter transgene (tdTomato including WPRE sequence). Genome annotation version 99. We generated 4 libraries. Each library was performed from a mammary carcinoma isolated from three independent mice. We profiled a total of 36,162 cells (T1:8,724 cells; T2:9580 cells; T3:8434 cells; T4:9424 cells). Libraries were sequenced to obtain more than 1.200 million reads in total (T1:>313M; T2:>313M; T3:>306M; T4:>308M). In all four libraries, mean read pairs per cell was above 30,000 (T1:35,878; T2:32,730; T3:36,377; T4:32,731). Confident mapping to exonic regions was higher than 61% for each library. For each library, median unique counts per cell was as follows: T1:1,922; T2:3,281; T3:2,854; T4:2,589; and median detected genes per cell was as follows: T1:842; T2:1,321; T3:1,142; T4:1,079.
Genome_build: GRCm38.99
Supplementary_files_format_and_content: Single-cell RNA-seq datasets. Each dataset contain three files: barcodes.tsv (a tab separated values file with cellular barcodes present in dataset); genes.tsv (a tab separated values file with IDs of quantified genes (Mouse ENSEMBL Gene ID and Gene Symbol); matrix.mtx (a matrix of count values, where rows are associated with the gene IDs above and columns correspond to the cellular barcodes). A standard or sparse matrix can be generated where genes.tsv file correspond to the genes or row names of the matrix, while barcodes.tsv corresponds to the cell or columns.
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| Submission date |
Oct 13, 2020 |
| Last update date |
May 09, 2024 |
| Contact name |
Angela Nieto |
| E-mail(s) |
anieto@umh.es
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| Organization name |
Instituto de Neurociencias (CSIC-UMH)
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| Department |
Developmental Neurobiology
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| Lab |
Cell plasticity in development and disease
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| Street address |
Av. Ramón y Cajal s/n
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| City |
San Juan de Alicante |
| State/province |
Alicante |
| ZIP/Postal code |
03550 |
| Country |
Spain |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE159478 |
Single-cell RNA profiling of mammary gland tumours in MMTV-PyMT mouse model of breast cancer. |
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| Relations |
| BioSample |
SAMN16429844 |
| SRA |
SRX9287117 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4830541_T1_barcodes.tsv.gz |
33.8 Kb |
(ftp)(http) |
TSV |
| GSM4830541_T1_genes.tsv.gz |
406.9 Kb |
(ftp)(http) |
TSV |
| GSM4830541_T1_matrix.mtx.gz |
25.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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