Comment[ArrayExpressAccession] E-MEXP-3402 Investigation Title Trancriptional regulation by axonal SMN on motor neuron cells Comment[Submitted Name] Trancriptional regulation by axonal SMN on motor neuron cells Comment[AEExperimentDisplayName] Transcription profiling by array of mouse motor neuron cells expressing human axonal SMN Comment[MIAMExpressLogin] maddalena Comment[MIAMExpressSubmissionID] 7863 Experiment Description Muscular atrophy (SMA) is an autosomal recessive disease causing selective motor neuron death by the loss of telomeric survival motor neuron gene, SMN1. Axonal SMN, a-SMN, is a truncated form of SMN, derived from an alternatively spliced SMN1 gene. (Setola, et. al. 2007 PNAS 104, 1959-1964). The cellular clones expressing a-SMN in a tetracycline-dependent manner were isolated from NSC34 by two-step stable transfection, first with the tetracycline-repressor construct and subsequently with the a-SMN cDNA. To identify novel a-SMN target genes, the transcriptome of several a-SMN clones was analyzed and compared with that of parental cells. Experimental Design in_vitro_design co-expression_design time_series_design cellular_process_design Comment[AEExperimentType] transcription profiling by array Experimental Factor Name CELL_LINE COMPOUND DOSE TIME GENOTYPE Experimental Factor Type cell_line compound dose time genotype Person Last Name Fratelli Person First Name Maddalena Person Email maddalena@marionegri.it Person Phone +39 02 3901 4217 Person Affiliation Istituto "Mario Negri" Person Address Molecular Biology, Via La Masa 19, Milano, Milano, 20156, Italy Person Roles submitter Person Roles Term Source REF MGED Ontology Quality Control Type biological_replicate Public Release Date 2012-01-07 Comment[ArrayExpressSubmissionDate] 2011-10-12 Publication Status not yet submitted Protocol Name P-MTAB-23340 P-MTAB-23341 P-MTAB-23342 P-MTAB-23343 P-MTAB-23344 Protocol Description Total RNA fraction was prepared using miRNeasy Mini Kit (Qiagen, Valencia, CA), and further purified and concentrated by RNeasy MinElute Cleanup Kit (Qiagen). The yield of RNA from one million cells was 20-30 microg. 1 and 2 are two independent experiments, A and B are technical replicates (two RNA samples extracted from duplicate culture wells).
(Parameters: Extracted product = total_RNA, Amplification = none) Images were analyzed using the Feature extraction software (Agilent, version 9.1. Data preprocessing was performed using variance stabilization normalization method (vsn, with the default settings) in the statistical programming and graphics environment R (http://cran.r-project.org). Processed data include single-sample generalized log (glog) of signal intensity from vsn. For microarray analysis, each cell clone for appropriate time points was prepared in duplicate. The RNA from each cell culture flask from the same experimental group was labeled, one with Cy3 and the other with Cy5 using Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion, Austin TX). The intensity of the fluorescent dye was approximately 100pmols/?microg of amino allyl RNA (aRNA), and 16 pmols each of the labeled aRNA was used for hybridization / well, using Whole Mouse Genome Oligo Microarray Kit (Agilent, Palo Alto, CA).
(Parameters: Amount of nucleic acid labeled = 1, Amplification = RNA polymerases, Mass unit = Micro gram) Vectors used were pcDNA6/TR and pcDNA4/TO from Invitrogen (Carlsbad, CA). NSC34 cells were transfected with pcDNA6/TR, and the TR4 clone was isolated by the resistance to Blasticidin S. The a-SMN cDNA fragment was inserted in HindIII/XbaI sites of pcDNA4/TO, and used for the subsequent transfection in TR4 cells. The a-SMN clones were selected for the resistance to both Blasticidine S and Zeocin. Individual clones were isolated by limiting dilution.

Induction of a-SMN was performed in the medium containing 1 microg/ml of tetracycline without any addition of other antibiotics. For RNA preparations, cells were grown to semi-confluence (500,000 cells/T25 flask) in the medium containing appropriate antibiotics, and then treated with tetracycline or without it (control) for 24, 56 or 72 hours. At the end of the treatment, cells were rinsed with pre-chilled PBS once, scraped in the presence of cold PBS, and harvested by centrifugation. The pelleted cells were kept frozen at -80 °C until use. The TR4 and a-SMN clones were maintained in the low-glucose (1g/l) DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 5% of Tet System-approved fetal calf serum (Clontech, Mountain View, CA). To obtain stable attachment of long axons of neuronal cells, the cells were grown in culture flasks pre-coated for one hour with Matrigel Matrix Basement Membrane (BD, Bedford, MA) diluted 50 times with DMEM. The TR4 clone was grown in the presence of 10 microg/ml

of Blasticidine S (Invitrogen), while a-SMN clones were cultured in the presence of 10 microg/ml of Blasticidine S and 50 microg/ml of Zeocin (Invitrogen).
(Parameters: time unit = seconds, min temperature = 37, temperature unit = C, media = low_glucose DMEM) Protocol Type nucleic_acid_extraction bioassay_data_transformation labeling specified_biomaterial_action grow Protocol Term Source REF MGED Ontology MGED Ontology MGED Ontology MGED Ontology MGED Ontology Term Source Name MGED Ontology ArrayExpress Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress SDRF File E-MEXP-3402.sdrf.txt