|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Nov 23, 2018 |
Title |
SS2_15_0048_J9 |
Sample type |
SRA |
|
|
Source name |
Mammary tumor fibroblast
|
Organism |
Mus musculus |
Characteristics |
strain: FVB/N-Tg(MMTVPyVT)634Mul/J age: 14 weeks marker genes: EpCAM-, CD45-, CD31-, NG2- tissue: Mammary tumor fibroblast cell type: cancer-associated fibroblasts (CAFs)
|
Extracted molecule |
total RNA |
Extraction protocol |
Breast tumors and mammary fat pads of FVB/N-Tg(MMTVPyVT)634Mul/J mice were collected after heart perfusion with PBS. Tumors were minced and digested in 10 ml FACS buffer PBS, 5% Cell dissociation buffer (Gibco), 0.2 % BSA containing 25 mg Collagenase II, 25 mg Collagenase IV, 5 mg DNAse, 15 min stirring at 37 °C. The digested cell suspension was strained through a 100 µm cell strainer with the plunger of a plastic syringe. After spinning down for 3 minutes at 300 x g, red blood cells were lysed using RBL buffer containing 0.15 M ammonium chloride and 10 mM sodium EDTA in ddH2O for 30 seconds. Red blood cell lysis was stopped with ice cold FACS buffer. Cells were counted after additional straining through a 70 µm mesh cells and centrifugation at 300 x g for 3 minutes. Fc regions on cells were blocked with 2 µl Fc-block in 50µl FACS buffer per 10^6 cells for 10 min on ice. The cells were incubated in staining cocktail containing anti-CD31-APC (1 µl/10^6 cells) anti-CD45-APC (1 µl/10^6 cells), anti-CSPG4-AF647 (0.4 µl/10^6 cells) and anti-CD326-APC (5 µl/10^6 cells) in FACS buffer for 30 minutes on ice. 4’-6’-diamidino-2-phenylindole (DAPI) was added to the cell suspension before sorting for alive EpCAM-, NG2-, CD31-, CD45- cells. The library were constructed following the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013).
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
|
|
Data processing |
Single-end 43 bp long reads were aligned at ESCG to mm10 mouse genome. RefSeq annotation was used for gene expression quantification, which resulted in 24490 endogenous gene counts and 92 spike-in counts The expression values were computed as reads per kilobase of gene model and million mappable reads (RPKMs) to normalize for varying sequencing depths across sequenced cells and the gene lengths. The expression values were computed per gene as described in Ramsköld et al. (2009 PLoS Comp Biol), using uniquely aligned reads and correcting for the uniquely alignable positions using MULTo (Storvall et al. 2013 PLoS ONE). Genome_build: mm10
|
|
|
Submission date |
Feb 28, 2018 |
Last update date |
Nov 23, 2018 |
Contact name |
Kristian Pietras |
E-mail(s) |
kristian.pietras@med.lu.se
|
Phone |
00462226429
|
Organization name |
Lund University
|
Department |
Laboratory Medicine
|
Lab |
Experimental Oncology
|
Street address |
Medicon Village, Building 404:A3
|
City |
Lund |
ZIP/Postal code |
22381 |
Country |
Sweden |
|
|
Platform ID |
GPL13112 |
Series (1) |
GSE111229 |
Single cell RNA-sequencing of EpCAM-, CD45-, CD31- NG2- murine mammary tumor fibroblasts |
|
Relations |
BioSample |
SAMN08620057 |
SRA |
SRX3750125 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
|