Comment[ArrayExpressAccession] E-MTAB-1192 Investigation Title Serge Nef Comment[Submitted Name] Serge Nef Experimental Design genetic_modification_design Comment[AEExperimentType] transcription profiling by array Comment[AEExperimentDisplayName] Transcriptional profiling by array of mouse wild-type and Insulin receptor (InsR)/Igf1 receptor (Igf1R) double knockout mutant Sertoli cells from testis at embryonic day E17.5 and Postnatal day 5 Experimental Factor Name developmental stage genotype Experimental Factor Type developmental stage genotype Person Last Name Nef Person First Name Serge Person Email Serge.Nef@unige.ch Person Address Serge Nef laboratory - CMU 1, rue Michel-Servet, 1211 Geneva GE, Switzerland Person Affiliation University Medical Center, University of Geneva Person Roles submitter Quality Control Type biological_replicate spike_quality_control Public Release Date 2013-03-09 Pubmed ID 23518924 Publication DOI 10.1210/me.2012-1258 Publication Author List Pitetti JL, Calvel P, Zimmermann C, Conne B, Papaioannou MD, Aubry F, Cederroth CR, Urner F, Fumel B, Crausaz M, Docquier M, Herrera PL, Pralong F, Germond M, Guillou F, Jégou B, Nef S Publication Title An essential role for insulin and IGF1 receptors in regulating sertoli cell proliferation, testis size, and FSH action in mice Experiment Description we performed the analysis of SOX9-GFP+ cells purified from mouse embryonic testis at embryonic day E17.5 and Postnatal day 5 and analyzed the effect of the deletion of the Insulin receptor (InsR) and Igf1 receptor (Igf1R) by comparing double mutant and control SOX9+ Sertoli cells Protocol Name P-MTAB-28273 P-MTAB-28274 P-MTAB-28275 P-MTAB-28276 P-MTAB-28277 P-MTAB-28278 Protocol Type growth nucleic_acid_extraction labeling hybridization image_acquisition bioassay_data_transformation Protocol Description 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. 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. 75ng of total RNA was reverse transcribed and amplified using the MessageAmpTM II-Biotin Enhanced Single Round RNA Amplification Kit (#1791, Ambion). 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). Microarrays were scanned with a GeneChip(r) scanner 3000 7G (Affymetrix). 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). Protocol Parameters Nucleic Acid Starting Amount Hybridization Concentration ; Hybridization Temperature ; Hybridization Time ; Hybridization Volume ; Labeled Nucleic Acid Amount Protocol Software SDRF File E-MTAB-1192.sdrf.txt