|
| Status |
Public on Dec 31, 2021 |
| Title |
Mouse Reprogrammed Mammary epithelial cells rep 4 |
| Sample type |
other |
| |
|
| Source name |
Mouse Reprogrammed Mammary epithelial cells rep 4
|
| Organism |
Mus musculus |
| Characteristics |
tissue: reprogrammed 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 |
other |
| 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 |
miRNA was labeled with Cy3-CTP using the miRCURY LNA microRNA power labeling kit (Exiqon, Inc, Woburn, MA), according to manufacturers’ protocol
|
| |
|
| Hybridization protocol |
Labeled RNA was hybridized to Agilent custom UCSF miRNA v3.4 multi-species 8x15K Ink-jet arrays (Agilent). Hybridizations were performed for 16 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 |
GPL13992 |
| Series (2) |
| GSE37308 |
In Vivo Organ Regeneration via Stroma-Dependent Adult Lineage Reprogramming (miRNA) |
| GSE37309 |
In Vivo Organ Regeneration via Stroma-Dependent Adult Lineage Reprogramming |
|
