|
| Status |
Public on Jul 23, 2019 |
| Title |
8362_CP6406902: Glast+ cells P7.2 |
| Sample type |
RNA |
| |
|
| Source name |
Glast (Slc1a3) positive cells isolated from developing retinas
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: retina
developmental stage: Postnatal Day 7 (P7)
cell type: Glast (Slc1a3) positive cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate Notch1+ cells and Glast+ cells, we employed an immunomagnetic cell separation approach using monoclonal biotin-conjugated anti-Notch1 and anti-Glast mouse antibodies and anti-biotin magnetic microbeads. RNA was extracted from cells using the Absolutely RNA Nanoprep kit (Agilent Technologies, Santa Clara, CA).
|
| Label |
Streptavidin-Alexa-647
|
| Label protocol |
Biotin-labeled complementary RNA was made from the total RNA according to Van Gelder’s protocol (Van Gelder RN, von Zastrow ME, Yool A, Dement WC, Barchas JD, et al. (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci U S A 87: 1663-1667.)
|
| |
|
| Hybridization protocol |
Biotinylated complementary RNA samples were fragmented, diluted in a formamide-containing hybridization buffer, and loaded on to the Mouse Exonic Evidence Based Oligonucleotide (MEEBO) microarray slides enclosed in custom hybridization chambers (for more information on the MEEBO oligonucleotide set please refer to http://alizadehlab.stanford.edu/). The slides were hybridized for 16-18 hours in a Model 400 hybridization oven (Scigene, Sunnyvale, CA).
|
| Scan protocol |
After hybridization, the microarray slides were washed under stringent conditions, stained with Streptavidin-Alexa-647 (Invitrogen, Carlsbad, CA), and scanned using an Axon GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA).
|
| Description |
We processed individual samples that each contained 200,000-500,000 cells.
|
| Data processing |
Spot intensities for each probe were calculated by subtracting median local background from median local foreground for each spot. The spot intensities were then normalized. After removing data for low quality spots, the mouse probes’ intensities were filtered to identify all probes with an intensity above a normalized threshold. For statistical analysis, microarray data were examined for differences by One-way ANOVA. Values of F > Fcrit. = 2.83 were designated as statistically significant.
|
| |
|
| Submission date |
Nov 08, 2018 |
| Last update date |
Jul 25, 2019 |
| Contact name |
Dmitry Ivanov |
| E-mail(s) |
divanov@med.miami.edu
|
| Phone |
305-482-4230
|
| Organization name |
University of Miami Miller School of Medicine
|
| Department |
Ophthalmology
|
| Street address |
1638 NW 10th Avenue
|
| City |
Miami |
| State/province |
FL |
| ZIP/Postal code |
33136 |
| Country |
USA |
| |
|
| Platform ID |
GPL25621 |
| Series (2) |
| GSE122301 |
Gene expression profiling of developing murine Müller glia |
| GSE122337 |
Development and epigenetic plasticity of murine Müller glia |
|
