Boudary cap cells were isolated from Krox20GFP(DT) heterozygous embryos at E12.5. The appropriate embryos were identified by GFP or YFP expression under fluorescent stereomicroscope (Leica, Nussloch, Germany) and head and viscera were eliminated. For boudary cap cell preparation, the meninges, carrying the nerve roots and dorsal root ganglion, were dissected from the neural tube from cervical to lumbo-sacral levels. Meninges from 3-5 litters were pooled and incubated at 37¡C for 20 min in 1 ml of HBSS (Sigma) with 0.05% trypsin (Invitrogen) and 3 mg/ml collagenase type III (Sigma). The digestion was stopped by addition of 0.1 ml of fetal calf serum (FCS), the material was washed 3 times with PBS, triturated, centrifuged at 200 g for 5 min, resuspended in HBSS and passed through nylon mesh of 20 µm pore diameter (Falcon) to get a single cell suspension. FACS was performed with a vantage flow cytometer (Becton-Dickinson, Mountain View, CA). Cells (1-2 x 106/ml) were analyzed for forward scatter, side scatter and GFP fluorescence with an argon laser (488 nm excitation, 520 nm emission). Dead cells and doublets were excluded by gating on forward and side scatter. GFP expressing cells were directly collected in the lysis buffer (Ambion) and stored at -20¡C until use.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the different purified cell populations using the RNAqueous-Micro kit (Ambion). The quality of the RNA was estimated using a Bioanalyser 2100 (Agilent). When required, amplification of the RNA (500 ng) was performed using the Amino Allyl MessageAmp aRNA kit (Ambion).
Label
Cy5
Label protocol
The labelling of the RNA (2-5 ug) was carried out by incorporation of amino-allyl dUTP.
Primary Schwann precursor cell cultures were obtained from E12.5 mouse embryos using a modification of a previously described protocol (Manent et al., 2003). Briefly, mouse embryo carcasses were dissected by decapitation and evisceration and trypsinized at 37¡C for 30 min. Dissociated cells were homogenized through a narrowed Pasteur pipette before centrifugation and resuspension in PM medium (DMEM, 10% FCS, 2 mM Forskolin, 10 ng/ml heregulin-b1 (HRG-§1, R&D Systems), 50 mg/ml Gentamycin, 2.5 µg/ml Fungizone. Cells were plated on PLL/laminin coated dishes (Sigma). After 48-72 h in culture, the cells were trypsinized and SCpr were sorted by an indirect magnetic cell sorting protocol based on SCpr specific expression of p75LNGFR (Manent et al., 2003). After sorting, p75-positive cells were replated in N2 medium (DMEM:F12 (1:1) supplemented with N2 complement, 2 mM Forskolin, 10 ng/ml HRG-§1 (R&D Systems), 50 mg/ml Gentamycin, 2.5 µg/ml Fungizone) on PLL/laminin coated dishes. Purity of the SCpr culture was assessed by immunofluorescence with antibodies against p75LNGFR (Chemicon).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the different purified cell populations using the RNAqueous-Micro kit (Ambion). The quality of the RNA was estimated using a Bioanalyser 2100 (Agilent). When required, amplification of the RNA (500 ng) was performed using the Amino Allyl MessageAmp aRNA kit (Ambion).
Label
Cy3
Label protocol
The labelling of the RNA (2-5 ug) was carried out by incorporation of amino-allyl dUTP.
Hybridization protocol
Microarrays were pre-hybridized during 1 hour at 42¡C in 5x SSC, 0.1% SDS, 1% BSA, washed with distilled water and dried. They were hybridized overnight at 42¡C in 25% formamide, 5x SSC, 0.1%SDS with Cy5- and Cy3-labelled cDNA, washed in 1x SSC, 0.2% SDS at 42¡C, in 0.1x SSC, 0.2% SDS and two times in 0.1x SSC at room temperature and finally were spin-dried.
Scan protocol
The hybridized microarrays were scanned using Genepix 4000B (Axon) and the resulting image files analyzed by GenePix Pro 6.0 software (Axon).
Description
Biological replicate one of two.
Data processing
Spots flagged by the GenePix software and saturating spots, where the median foreground intensity is greater than 60,000 in one of the two channels, were discarded from the result files. The data were normalized using Goulphar (Lemoine et al.,2006) applying a global Lowess correction followed by a print-tip median normalization step.
