|
| Status |
Public on Oct 30, 2019 |
| Title |
594_50: WT-MG-NeuT |
| Sample type |
SRA |
| |
|
| Source name |
Mammary Gland
|
| Organism |
Mus musculus |
| Characteristics |
strain background: Balb/c
genotype: WT
tissue: Mammary Gland
|
| Treatment protocol |
Mammary glands or mammary tumors (size±225mm2), spleen and bone marrow were harvested from 4 month old K14cre;Cdh1F/F;Trp53F/F female mice containing early lesions, 8 months old tumor-bearing K14cre;Cdh1F/F;Trp53F/F, 4 month old tumor-bearing MMTV-NeuT female mice and age- and sex-matched FVB/N and Balb/c mice. Tumors and mammary glands were mechanically chopped with a McIlwain Tissue Chopper (Ted Pella, Inc, CA, USA) and were enzymatically digested for 1 hour at 37°C with 3 mg/ml collagenase type A (Roche) and 1.5 mg/ml porcine pancreatic trypsin (BD Biosciences) in serum-free DMEM medium. Digestion was stopped by the addition of DMEM supplemented with 8% FBS and the suspension was disaggregated through a 70 μm cell strainer. Spleens and bone marrow cells from tibiae and femora were harvested and disaggregated through a 70 μm cell strainer. Splenic suspensions were subsequently treated twice with NH4Cl erythrocyte lysis buffer for 3 min at room temperature. All single-cell suspensions were stained for 20 min at 4°C in the dark with anti-mouse F4/80 (1:200; BM8; eBioscience) and anti-mouse CD11b (1:200; M1/70; eBioscience) in IMDM supplemented with 2% FBS, 0.5% beta-mercaptoethanol, 0.5mM EDTA, Pen/Strep. Cells were then washed and incubated with magnetic anti-APC MicroBeads (Miltenyi Biotec) following the manufacturer’s guidelines. Isolation of F4/80+ cells from the CD11b+-enriched fraction was performed on a BD FACS ARIA II sorter with Diva software (BD Biosciences).
|
| Growth protocol |
The generation and characterization of K14cre;Cdh1F/F;Trp53F/F mice have been previously described in detail. The mice were backcrossed onto the FVB/N background and genotype was confirmed by PCR. MMTV-NeuT mice on a Balb/c background were purchased from Charles River Laboratories and were bred in house. Female K14cre;Cdh1F/F;Trp53F/F and MMTV-NeuT mice were monitored twice every week for the onset of mammary tumor formation by caliper and palpation measurement starting at 2 or 4 months of age, respectively. Mice were kept in open cages with food and water provided ad libitum at a 12-hour light/dark cycle.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini and Micro Kits (Qiagen)
Ovation RNA-Seq system V2 and Encore Rapid library systems protocols (NuGEN)
50 bp single-end run was performed on a HiSeq1500
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
base calling and de-multipexing using CASAVA version 1.8
Alignment withTopHat2 version v2.0.11 and Bowtie2 version 2.2.1 using the default parameters
Quantification with Partek Genomics Suite V6.6
reads mapped to each transcript against the RefSeq mm10 annotation download on May 2015
Normalization using DeSeq2
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include DeSeq2 normalized counts for each Sample.
|
| |
|
| Submission date |
Feb 08, 2019 |
| Last update date |
Oct 30, 2019 |
| Contact name |
Joachim Schultze |
| E-mail(s) |
j.schultze@uni-bonn.de
|
| Organization name |
LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)
|
| Department |
Genomics and Immunoregulation
|
| Street address |
Carl-Troll-Strasse 31
|
| City |
Bonn |
| State/province |
NRW |
| ZIP/Postal code |
53115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18480 |
| Series (1) |
| GSE126268 |
Transcriptional signatures derived from murine tumor-associated macrophages predict outcome in breast cancer patients |
|
| Relations |
| BioSample |
SAMN10888668 |
| SRA |
SRX5351974 |
