|
| Status |
Public on Dec 31, 2021 |
| Title |
Mouse Mammary epithelial cells rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse Mammary epithelial cells rep1
|
| Organism |
Mus musculus |
| Characteristics |
tissue: mammary
cell type: epithelial cells
|
| Growth protocol |
To prepare embryonic lung epithelium, noon of the day when a vaginal plug was detected was considered approximately E0.5. Embryos were collected in cold 1 X phosphate buffered saline (PBS). Lungs were dissected out from embryos at the E13.5 stages. Epithelium and mesenchyme were separated as reported previously86. For adult donor cell preparation, female mice of 10-weeks of age were sacrificed and perfused with PBS. Lungs and mammary glands were harvested, minced and dissociated in buffer [5% fetal bovine serum (FBS), insulin 5 µg/ml, Penicillin-Streptomycin 100 U/ml in DMEM/F12] containing trypsin (2 mg/ml) and collagenase A (2 mg/ml) for 45 minutes and 30 minutes, respectively, at 37ºC. Primary epithelia were purified by differential centrifugation: specifically, lung and mammary epithelia were washed in dissociation buffer without collagenase and collected using a swing-bucket centrifuge at 400 X g. The process was repeated for seven and five times for lung and mammary tissues, respectively. To prepare single cells, primary epithelia from embryonic lung and adult lung and mammary gland were washed in Hank’s (Ca2+ Mg2+ free) buffer and dissociated in 0.05% Trypsin/EDTA buffer. Cells were counted using a Hausser Scientific Hemocytometer and prepared in 50% Matrigel™ (growth factor reduced, BD Biosciences) for transplantation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sample preparation, labeling, and array hybridizations were performed according to standard protocols from the UCSF Shared Microarray Core Facilities and Agilent Technologies. Total RNA quality was assessed using a Pico Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy3
|
| Label protocol |
mRNA was amplified and labeled with Cy3-CTP using the Agilent low RNA input fluorescent linear amplification kits following the manufacturers protocol (Agilent). Labeled cRNA was assessed using the Nandrop ND-100 (Nanodrop Technologies, Inc., Wilmington DE).
|
| |
|
| Hybridization protocol |
Equal amounts of Cy3 labeled target were hybridized to Agilent whole mouse genome 4x44K Ink-jet arrays (Agilent). Hybridizations were performed for 14 hrs, according to the manufacturers protocol (Agilent).
|
| Scan protocol |
Arrays were scanned using the Agilent microarray scanner (Agilent).
|
| Data processing |
Raw signal intensities were extracted with Feature Extraction v10.1 software (Agilent)
|
| |
|
| Submission date |
Apr 16, 2012 |
| Last update date |
Dec 31, 2021 |
| Contact name |
Andrew H Sims |
| E-mail(s) |
andrew.sims@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
Institute of Genetics and Molecular Medicine
|
| Lab |
Applied Bioinformatics of Cancer
|
| Street address |
Systems Medicine Building
|
| City |
Carrington Crescent |
| State/province |
Edinburgh |
| ZIP/Postal code |
EH4 2XR |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE37289 |
In Vivo Organ Regeneration via Stroma-Dependent Adult Lineage Reprogramming (mRNA) |
| GSE37309 |
In Vivo Organ Regeneration via Stroma-Dependent Adult Lineage Reprogramming |
|
