Illumina HiSeq 2000 sequencing; GSM3025845: SS2_15_0048_A1; Mus musculus; RNA-Seq

SS2_15_0048_A1

This study was conducted to determine heterogeneity of cancer-associated fibroblasts (CAFs) in mammary tumors, by unsupervised analysis of single cell transcriptomes. Overall design: 768 single EpCAM-, CD45-, CD31- NG2- fibroblasts were isolated from mammary tumors of two 14 week old MMTV-PyMT mice. The cells were sequenced following the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013).

Single cell RNA-sequencing of EpCAM-, CD45-, CD31- NG2- murine mammary tumor fibroblasts

Submitted by Gene Expression Omnibus on 24-NOV-2018

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).

Single cell RNA-sequencing of EpCAM-, CD45-, CD31- NG2- murine mammary tumor fibroblasts

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