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Sample GSM3025862

Query DataSets for GSM3025862

Status

Public on Nov 23, 2018

Title

SS2_15_0048_A18

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