B6D2 wild type and heterozygote Krox20GFP(DT)/+ (Vermeren al 2003) embryos were used for dorsal root ganglion (DRG) and Schwann Cells (SC) cultures respectively. DRG cultures: DRG were dissected from E17.5 embryos. After dissociation for 10 min at 37°C on a rocking table in Ca and Mg-free HBSS containing collagenase type I (130 U/ml), dispase I (2.5 mg/ml), followed by mechanical dissociation, cells were resuspended in DRG medium: DMEM supplemented with L-glutamin (2 mM), Horse serum (5%) and 2.5s NGF (50 ng/ml) (BD Biosciences), and plated onto 60 mm diameter dishes or 12 mm diameter CML coverslips that were coated with poly-L-lysine (PLL, 10 µg/ml, Sigma) and rat collagen type I (5 µg/cm2, Sigma). After 1 day, cytosine arabinoside (10 µM) was added for 2 days to the medium to get rid of non neuronal cells and cells were then maintained in DRG medium. SC cultures: Sciatic nerves were dissected from E17.5 embryos into ice-cold Ca, Mg-free HBSS and dissociated by incubating for 20 min at 37°C on a rocking table in Leibovitz medium (L15, Gibco) containing collagenase type I (130 U/ml) (Gibco), dispase I (2.5 mg/ml) (Roche), followed by mechanical dissociation. Cells were plated onto PLL and collagen coated dishes, and maintained in SC medium : 1:1 ratio of DMEM and Ham’s F12 (Gibco), 10% Fetal Calf Serum (FCS) supplemented with Na-selenite (0.5 ng/ml) (Sigma), progesterone (0.63 ng/ml) (Sigma), putrescine (1.6 µg/ml) (Sigma), transferring (0.1 mg/ml) (Sigma), insulin (0.5 µg/ml) (Sigma), forskolin (2 µM) (Sigma), heregulin (10 ng/ml) (Sigma), gentamycin (50 µg/ml), fungizone (2.5 µg/ml). Indirect magnetic cell sorting: The mouse SC purification was performed using the technique of magnetic cell separation based on SC membrane expression of the low affinity Nerve Growth Factor receptor (p75NGFR) (Manent et al, 2003) according to manufacturer’s instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Purity of SCs after magnetic cell sorting was quantified after immunolabelling as the percentage of p75NGFR- and S100- positive cells with respect to the total of cells counted. At least 100 cells per condition were counted and experiments were repeated 11 times. Results were expressed as mean ± SD, and statistical analysis was performed using Student’s t test. The purified population (enriched to 97 ± 2 % of SC, n=11) was either plated on coated dish or seeded onto DRG neurons and cocultures were then maintained for 1 to 4 days. Fluorescence activated cell sorting (FACS). Cells from SC-DRG cocultures were collected by trypsinisation and kept on ice in 0.1 M PBS containing 0,2% FCS. FITC fluorescent cells (GFP+ SCs) were sorted using a MoFlow cytometer (Becton Dickinson) and collected in lysis buffer (Ambion) to start the RNA extraction and to prevent any RNA degradation. An aliquot of cells isolated by FACS was preserved in 0.1 M PBS 0,2% FCS and cells were seeded for a couple of hours to allow them to attach to check cell sorting efficiency by immunocytochemistry using an anti-GFP antibody.
Extracted molecule
polyA RNA
Extraction protocol
mRNA was extracted using the micro scale RNA isolation kit (Ambion). Amplification of RNA samples for microarray experiments was achieved using the Amino Allyl MessageAmpTM aRNA kit (Ambion). Quantity and integrity of the RNA were measured with the RNA 6000 nano assay kit by the Agilent 2100 Bioanalyser (Agilent Technologies, Germany). All kits were used according to the manufacturer’s instructions.
Label
Cy3
Label protocol
The labelling of the RNA (5 ug) was carried out by incorporation of amino-allyl dUTP.
cell type: Schwann cells cocultured with neurons tissue: embryo
Treatment protocol
B6D2 wild type and heterozygote Krox20GFP(DT)/+ (Vermeren al 2003) embryos were used for dorsal root ganglion (DRG) and Schwann Cells (SC) cultures respectively. DRG cultures: DRG were dissected from E17.5 embryos. After dissociation for 10 min at 37°C on a rocking table in Ca and Mg-free HBSS containing collagenase type I (130 U/ml), dispase I (2.5 mg/ml), followed by mechanical dissociation, cells were resuspended in DRG medium: DMEM supplemented with L-glutamin (2 mM), Horse serum (5%) and 2.5s NGF (50 ng/ml) (BD Biosciences), and plated onto 60 mm diameter dishes or 12 mm diameter CML coverslips that were coated with poly-L-lysine (PLL, 10 µg/ml, Sigma) and rat collagen type I (5 µg/cm2, Sigma). After 1 day, cytosine arabinoside (10 µM) was added for 2 days to the medium to get rid of non neuronal cells and cells were then maintained in DRG medium. SC cultures: Sciatic nerves were dissected from E17.5 embryos into ice-cold Ca, Mg-free HBSS and dissociated by incubating for 20 min at 37°C on a rocking table in Leibovitz medium (L15, Gibco) containing collagenase type I (130 U/ml) (Gibco), dispase I (2.5 mg/ml) (Roche), followed by mechanical dissociation. Cells were plated onto PLL and collagen coated dishes, and maintained in SC medium : 1:1 ratio of DMEM and Ham’s F12 (Gibco), 10% Fetal Calf Serum (FCS) supplemented with Na-selenite (0.5 ng/ml) (Sigma), progesterone (0.63 ng/ml) (Sigma), putrescine (1.6 µg/ml) (Sigma), transferring (0.1 mg/ml) (Sigma), insulin (0.5 µg/ml) (Sigma), forskolin (2 µM) (Sigma), heregulin (10 ng/ml) (Sigma), gentamycin (50 µg/ml), fungizone (2.5 µg/ml). Indirect magnetic cell sorting: The mouse SC purification was performed using the technique of magnetic cell separation based on SC membrane expression of the low affinity Nerve Growth Factor receptor (p75NGFR) (Manent et al, 2003) according to manufacturer’s instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Purity of SCs after magnetic cell sorting was quantified after immunolabelling as the percentage of p75NGFR- and S100- positive cells with respect to the total of cells counted. At least 100 cells per condition were counted and experiments were repeated 11 times. Results were expressed as mean ± SD, and statistical analysis was performed using Student’s t test. The purified population (enriched to 97 ± 2 % of SC, n=11) was either plated on coated dish or seeded onto DRG neurons and cocultures were then maintained for 1 to 4 days. Fluorescence activated cell sorting (FACS). Cells from SC-DRG cocultures were collected by trypsinisation and kept on ice in 0.1 M PBS containing 0,2% FCS. FITC fluorescent cells (GFP+ SCs) were sorted using a MoFlow cytometer (Becton Dickinson) and collected in lysis buffer (Ambion) to start the RNA extraction and to prevent any RNA degradation. An aliquot of cells isolated by FACS was preserved in 0.1 M PBS 0,2% FCS and cells were seeded for a couple of hours to allow them to attach to check cell sorting efficiency by immunocytochemistry using an anti-GFP antibody.
Extracted molecule
polyA RNA
Extraction protocol
mRNA was extracted using the micro scale RNA isolation kit (Ambion). Amplification of RNA samples for microarray experiments was achieved using the Amino Allyl MessageAmpTM aRNA kit (Ambion). Quantity and integrity of the RNA were measured with the RNA 6000 nano assay kit by the Agilent 2100 Bioanalyser (Agilent Technologies, Germany). All kits were used according to the manufacturer’s instructions.
Label
Cy5
Label protocol
The labelling of the RNA (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 (Molecular Devices, Sunnyvale, CA, USA) and the resulting image files analyzed by GenePix Pro 5.0 software (Axon).
Description
Biological replicate one of three.
Data processing
For each GenePix output file, two filters were applied, one to clear out spots and another one to discard saturating spots where the median foreground intensity was greater than 60,000 in one of the two channels. The resulting median foreground intensities were normalized, without background signal subtraction, using a global Lowess correction followed by a print-tip median normalization step (Lemoine et al., 2006).
