Microarrays were scanned with a GeneChip(r) scanner 3000 7G (Affymetrix).
For each probe, 20 ug of the amplified biotinylated cRNA was fragmented and hybridized to Mouse Genome 430 2.0 Arrays (Affymetrix, High Wycombe, UK) as previously described (Cederroth et al., 2007, http://www.sciencedirect.com/science/article/pii/S0012160608013572).
75ng of total RNA was reverse transcribed and amplified using the MessageAmpTM II-Biotin Enhanced Single Round RNA Amplification Kit (#1791, Ambion).
For RNA extraction, GFP+ or GFP- cells were directly sorted in lysis buffer from Qiagen RNeasy kit and extracted using RNeasy micro kit from Qiagen according to the manufacturer's protocol. RNA integrity and quantity were assessed using RNA 6000 picochips with an Agilent 2100 bioanalyzer. To minimize the effects of biological variability, three independent sets of total RNA per condition were isolated from a pool of GFP+ cells originating from >=3 males and used as a template for probe generation.
At relevant stages, control and SC-Insr;Igf1r male pups homozygous for Sox9:EGFP were sacrificed (n=5-10). GFP positive testes were decapsulated and incubated for 10 minutes in 0.5% trypsin EDTA (Gibco) and RQ1/DNAse (4 mg/ml) at 37 degrees C. To ensure a complete cellular dissociation, an additive digestive step is performed by incubating the cell clusters in a solution/cocktail of collagenase (10 mg/ml), hyaluronidase (20 mg/ml) and DNAse (4 mg/ml). To generate a single cell suspension, PBS w/o Ca2+/Mg2+ was added and the preparation was filtered through a 40 ?m nylon cell strainer. GFP+ cells were sorted using a FacsVantage SE (BecktonDickinson). See Nel-Themaat et al., 2009 for more details.
CEL files were uploaded into the AMEN v.1.3.4 software (Chalmel, 2008) (http://sourceforge.net/project/AMEN) and submitted to the Robust Multi-array Average (RMA) procedure allowing for summarization of probeset intensities, background correction and data normalization (Bolstad et al., 2003).