To isolate Notch1+ cells we employed an immunomagnetic cell separation approach using monoclonal biotin conjugated anti-Notch1 mouse antibodies and anti-biotin magnetic microbeads.
Label
Streptavidin-Alexa-647
Label protocol
RNA samples were extracted from whole retina and Notch1+ cells using the Absolutely RNA® Nanoprep kit (Agilent Technologies, Santa Clara, CA). Biotin-labeled complementary RNA was made from 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
Sample name: E14_19709353 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 intensity above a normalized threshold. For statistical analysis, microarray data were examined for differences by One-way ANOVA or Student's t-test. Values of P < 0.05 were designated as statistically significant.
