Investigation Title Transcription profiling of synectin knock-out mouse primary endothelial cells before and after FGF2 induction Comment[Submitted Name] Simons Murine Synectin SSH_Array Study Experimental Design stimulus_or_stress_design cellular_modification_design dye_swap_design genetic_modification_design transcription profiling by array Experimental Design Term Source REF mo mo mo EFO Comment[ArrayExpressReleaseDate] 2006-10-01 Comment[AEMIAMESCORE] 5 Comment[ArrayExpressAccession] E-MEXP-820 Comment[MAGETAB TimeStamp_Version] 2010-08-11 16:02:29 Last Changed Rev: 13058 Experimental Factor Name compound dose genotype Experimental Factor Type compound_treatment_design dose genotype Experimental Factor Term Source REF Person Last Name Smith Schwartz Mulvihill Chittenden Simons Lanahan Person First Name Kimberly Stephen Eileen Thomas Michael Anthony Person Mid Initials W A Person Email thomas.w.chittenden@dartmouth.edu Person Phone 6036502633 Person Fax 6036530510 Person Address 546W, Borwell, One Medical Center Drive Person Affiliation University of Washington School of Medicine University of Washington School of Medicine University of Washington School of Medicine Medicine dartmouth Medical School dartmouth Medical School Person Roles submitter Person Roles Term Source REF Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2006-10-01 PubMed ID 16940428 Publication DOI 16940428 Publication Author List Anthony Lanahan; Thomas Chittenden; Eileen Mulvihill; Kimberly Smith; Stephen Schwartz; Michael Simons Publication Title Synectin-dependent gene expression in endothelial cells Publication Status journal_article Publication Status Term Source REF Experiment Description Transcriptional regulatory effects of synectin deletion in murine primary endothelial cells both before and after FGF2 induction. Protocol Name P-MEXP-30059 P-MEXP-30054 P-MEXP-30204 P-MEXP-30206 P-MEXP-30205 P-MEXP-30089 P-MEXP-30111 P-MEXP-30122 Protocol Type specified_biomaterial_action grow nucleic_acid_extraction labeling labeling hybridization feature_extraction bioassay_data_transformation Protocol Description SSH and Generation of Subtracted Libraries:
SSH was performed with the PCR-SelectTM cDNA Subtraction Kit (Clontech). Starting material consisted of 2ug poly(A+) RNA from FGF2 treated (1/2, 1, 2 hours) synectin+/+ and -/- lung endothelial cells. cDNA was synthesized, digested with RsaI and ligated with two different adapters. Subtracted libraries were generated using adapted synectin+/+ cDNA subtracted against synectin-/- mRNA and adapted synectin-/- cDNA subtracted against synectin-+/+ mRNA. The first round of hybridization was performed at 68oC for 8 hours and the second round of hybridization was incubated at 68C for 22 hours. After two rounds of subtractive hybridization, two rounds of PCR were performed to amplify differentially expressed cDNA fragments. The PCR products were digested with NotI/EagI, cloned into pBluescript(SK) and transformed into E. Coli.

Colony PCR and Differential Screening:
A portion of each SSH library was plated on LB ampicillin plates with X-GAL and “white” insert containing colonies were picked at random for further screening. Individual colonies were picked, gridded onto an LB ampicillin plate and then the remaining bacteria were used for colony PCR using the M13 forward and reverse primers. PCR conditions were: 95C/10minutes, 40X (94C/30sec., 64C/1min., 72C/2min), 72C/5 min. PCR products were denatured, neutralized and slot blotted onto duplicate nitrocellulose filters. Filters were prehybridized overnight at 65C in 5X SSC, 5X Denhardt’s solution, 50mM NaPO4, 10mM EDTA, 0.2% SDS and 100ug/ml sheared sonicated salmon sperm DNA. 32P-dCTP labeled double stranded cDNA probes were prepared from synectin-/- and +/+ cDNA by random nonamer labeling (Stratagene). Probes were denatured, added to freshly made hybridization buffer and the filters hybridized for 40 hours. Filters were washed twice at room temperature with 2X SSC/0.2% SDS, twice at 65C with 0.5X SSC/0.2% SDS and exposed to X-Ray film. After exposure, clones showing differential hybridization to wildtype or knockout cDNA were expanded for further analysis. Primary endothelial cells from adult mice hearts and lungs were isolated by standard dissection techniques. Briefly, the hearts and lungs of 4 mice (both Synectin Ko and Wt control) were harvested, minced finely with scissors, and then digested in 25ml collagenase 0.2% (w/v) at 37C for 45 min. The crude cell preparation was pelleted and resuspended in DPBS. The cell suspension was incubated with PECAM-1-coated beads (IgG Dynal beads from Dynal Corp., Great Neck, NY) at room temperature for 10min. The bead-bound cells were recovered, washed with DMEM-20%FBS, suspended in 12ml complete culture medium (DMEM containing 20% fetal calf serum, 100ug/ml heparin, 100ug/ml ECGF growth supplement), and then plated in a gelatin-coated 75cm2 tissue culture flask. Cells were cultured at 37C. Endothelial cell poly A+ RNA was extracted with an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. SSH was performed with the PCR-SelectTM cDNA Subtraction Kit (Clontech). Starting material consisted of 2ug poly(A+) RNA from basal and FGF2 treated (1/2, 1, 2 hours) synectin+/+ and -/- lung endothelial cells. cDNA was synthesized, digested with RsaI and ligated with two different adapters. Subtracted cDNA was generated using adapted synectin+/+ cDNA subtracted against synectin-/- mRNA and adapted synectin-/- cDNA subtracted against synectin-+/+ mRNA. The first round of hybridization was performed at 68C for 8 hours and the second round of hybridization was incubated at 68C for 22 hours. After two rounds of subtractive hybridization, two rounds of PCR were performed to amplify differentially expressed cDNA fragments. PCR products from the subtraction of KO cDNA against an excess of WT mRNA were labeled with Cy3, while PCR products from the subtraction of WT cDNA against an excess of KO mRNA were labeled with Cy5 using random priming and Exo-Klenow kit (Invitrogen) according to manufacture’s recommendations. Dye swapping was performed to control for labeling bias. PCR products from the subtraction of KO cDNA against an excess of WT mRNA were labeled with Cy3, while PCR products from the subtraction of WT cDNA against an excess of KO mRNA were labeled with Cy5 using random priming and Exo-Klenow kit (Invitrogen) according to manufacture’s recommendations. Dye swapping was performed to control for labeling bias. cDNA probes were immobilized to the surface of the slide by baking the array for 3 hours at 80C. Slides were blocked for 45 minutes in a 42C solution of 5xSSC, 1% BSA, and 0.1% SDS. Prepared slides were hybridized overnight at 45C with the labeled sample in a 50% formamide hyb buffer (50% formamide, 5xSSC, 5xDenhardt’s reagent, 0.1% SDS, 100ug/ml Cot I DNA, 20ug/ml poly A). Slides were washed in decreasing concentrations of SSC and SDS. Dye swapping was performed on a second cDNA chip to control for labeling bias. The slides were scanned with an Axon GenePix 4000a scanner and the images were analyzed using Silicon Genetics GeneSpring software by manufacture's recommendations. All slides were normalized using a LOWESS function to address intensity-dependent dye biases. The data from the dye flip experiments were merged and an Excel spreadsheet report created including the normalized ratio of synectin -/- vs synectin +/+, the treatment average for each spot, the control average for each spot, the t-test p value of each synectin -/- vs synectin +/+ spot and the annotation. Protocol Parameters max temperature;min temperature; Extracted product;Amplification; Label used;Amplification; Amplification;Label used; temperature;Chamber type;Quantity of label target used; Protocol Hardware GenePix Professional 4200A [Axon Instruments] Protocol Software GenePix 4100A [Axon Instruments] Protocol Contact Protocol Term Source REF mo The MGED Ontology The MGED Ontology SDRF File E-MEXP-820.sdrf.txt Term Source Name mo atcc ncbitax The MGED Ontology ArrayExpress mo EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.atcc.org/ http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version