|
| Status |
Public on Jun 16, 2012 |
| Title |
Mouse_colon_BEC_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse, colon, normal, blood endothelial cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
gender: female
age: 8 weeks
tissue: colon
cell type: blood vascular endothelial cells
condition: normal, healthy
cell isolation method: FACS
|
| Treatment protocol |
Longitudinally opened mouse colons were washed in cold PBS and the mucus was gently scraped off in 1 mM DTT for 3 min. Colon pieces were incubated at 370C in 8 mg/ml collagenase IV (Invitrogen), 0.5 mg/ml DnaseI (Roche), and 5 mM CaCl2 in PBS for 15 min while rotating. Tissue suspensions were passed through a 70 µm cell strainer (BD Biosciences) while flushing with 2% FBS in PBS. Cell suspensions were centrifuged at 500 g for 10 min at RT and resuspended in PBS containing 2% FBS and 1 mM EDTA. The following antibodies were used for FACS: APC-conjugated rat anti-mouse CD31; FITC-conjugated rat anti-mouse CD45.2 (BD Biosciences); hamster anti-mouse podoplanin (clone 8.1.1) followed by anti-hamster PE-conjugated secondary antibody (Invitrogen); and isotype control antibodies (BD Biosciences). FACS was performed using a FACSAria and the FACSDiva software (BD Biosciences).
|
| Growth protocol |
Mice were maintained under conventional conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells from mouse colon tissue were FACS sorted directly into RLT Plus lysis buffer (Qiagen) supplemented with β-mercaptoethanol at 4C, for immediate cell lysis and RNA preservation. Cells were lysed by vortexing for 1 min. RNeasy Micro Plus Kit (Qiagen) was used to extract total RNA according to manufacturer’s instructions. Note: gDNA eliminator column was used to eliminate genomic DNA. Extracted RNA was amplified using the Whole Transcriptome-Ovation Pico RNA Amplification System (NuGEN Technologies) according to the manufacturer’s instructions. Briefly, the primers had a DNA portion that hybridizes either to the 5’ portion of the poly (A) sequence or randomly across the transcript. SPIA amplification, a linear isothermal DNA amplification process, was used to prepare single-stranded cDNA in the antisense direction of the mRNA starting material. The quantity and quality of amplified cDNA was determined with a NanoDrop ND 1000 (NanoDrop Technologies) and a Bioanalyzer 2100 (Agilent).
|
| Label |
biotin
|
| Label protocol |
Single-stranded cDNA (4 μg) was fragmented and biotin-labeled using FL-Ovation cDNA Biotin Module V2 kit (NuGEN Technologies) according to the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
Biotin-labeled cDNA targets were mixed in 220 μl of Hybridization Mix (Affymetrix Inc.) containing hybridization controls and control oligonucleotide B2 (Affymetrix Inc.). Samples were hybridized to GeneChip Mouse Genome 430 2.0 arrays for 18h at 45°C. Arrays were washed using an Affymetrix Fluidics Station 450 FS450 0004 protocol.
|
| Scan protocol |
An Affymetrix GeneChip Scanner 3000 was used to measure the fluorescence intensity emitted by the labeled target.
|
| Description |
Extracted RNA was used in linear isothermal whole transcriptome amplification (SPIA amplification) that results in single-stranded cDNA.
BEC_3
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) as implemented in the BioConductor simpleaffy package using default parameters, in particular the trimmed mean target intensity of each array was set to 100. The data values in the Sample data table are Signal Log2 Values, as described in the Affymetrix Statistical Algorithms Description Document. They are calculated as Tukey bi-weight values from logged probe values. The chip description file used for summarization was the standard Affymetrix Mouse430_2 CDF file.
|
| |
|
| Submission date |
May 27, 2010 |
| Last update date |
Jun 16, 2012 |
| Contact name |
Giorgia Jurisic |
| Organization name |
ETH Zurich
|
| Department |
Institute of Pharmaceutical Sciences
|
| Street address |
Wolfgang-Pauli Strasse 10
|
| City |
Zurich |
| ZIP/Postal code |
8093 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE22034 |
Transcriptional profiling of ex vivo isolated mouse blood vascular and lymphatic endothelial cells |
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