|
| Status |
Public on Aug 12, 2014 |
| Title |
MTECs_ALId2vd7_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
post-ALI MTECs: day 2, replicate #1
|
| Organism |
Mus musculus |
| Characteristics |
cell type: tracheal epithelial cell
culture conditions: cells were cultured at air-liquid interface (ALI)
time (postali_day): 2
strain: C57BL/6J
gender: Male
|
| Growth protocol |
Primary-culture mouse tracheal epithelial cells (mTECs) were established on Transwell membranes using air-liquid interface (ALI) conditions as described previously (You et al, AJP Lung, 2002, PMID 12388377)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy micro kit (Qiagen, Valencia, CA)
|
| Label |
Cy3
|
| Label protocol |
From total RNA, first strand cDNA was generated by fluorophore/dendrimer specific oligo-dT primed reverse transcription (Superscript II; Invitrogen) utilizing the 3DNA Array 900 kit (Genisphere). The 3DNA dT primers bear a capture sequence on the 5′ end that during hybridization binds Genisphere dendrimers labeled with either Cy3 or Cy5 dye in a sequence dependent fashion. Each sample was split in half, and labeled with each dye separately, creating technical replicates for each condition.
|
| |
|
| Channel 2 |
| Source name |
post-ALI MTECs: day 7, replicate #1
|
| Organism |
Mus musculus |
| Characteristics |
cell type: tracheal epithelial cell
culture conditions: cells were cultured at air-liquid interface (ALI)
time (postali_day): 7
strain: C57BL/6J
gender: Male
|
| Growth protocol |
Primary-culture mouse tracheal epithelial cells (mTECs) were established on Transwell membranes using air-liquid interface (ALI) conditions as described previously (You et al, AJP Lung, 2002, PMID 12388377)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy micro kit (Qiagen, Valencia, CA)
|
| Label |
Cy5
|
| Label protocol |
From total RNA, first strand cDNA was generated by fluorophore/dendrimer specific oligo-dT primed reverse transcription (Superscript II; Invitrogen) utilizing the 3DNA Array 900 kit (Genisphere). The 3DNA dT primers bear a capture sequence on the 5′ end that during hybridization binds Genisphere dendrimers labeled with either Cy3 or Cy5 dye in a sequence dependent fashion. Each sample was split in half, and labeled with each dye separately, creating technical replicates for each condition.
|
| |
|
| |
| Hybridization protocol |
Two hybridizations were carried out in a sequential manner. The primary hybridization was performed by adding cDNA to the microarray which was incubated at 63 °C for 16-20 h. A second hybridization was carried out using the Cy3 and Cy5 labeled fluorescent dendrimers (Genisphere) which contain oligos complementary to the capture sequence in the dT primers.
|
| Scan protocol |
Slides were scanned immediately after hybridization in a PerkinElmer ScanArray Express HT scanner to detect Cy3 and Cy5 fluorescence. Each slide was scanned twice at constant laser power with two different PMT settings. Each scan was exported in GenePix format. The scans at both settings were combined using functional regression estimators to enhance detection of differential expression (see PMID 19058173).
|
| Description |
post-ALI MTECs: day 2 versus day 7, replicate #1
|
| Data processing |
Microarray normalization and statistical analysis is performed using packages from the Bioconductor project executed in the R programming environment (PMID 15461798). Scans at both settings were combined using functional regression estimators generating a single dataset for each array (PMID 19058173). Within array normalization was performed using the robustspline method, and between arrays normalization was performed using the Aquantile method as implemented in the LIMMA package (PMID 16646809). Differential expression was assessed using linear models and empirical Bayes moderated F statistics, also using the LIMMA package, using a single model for time course data (PMID 15738394). As interpretation of microarray data is improved through the use of multiple annotation sources (PMID 20089164), probes were annotated using the manufacturer’s annotation files (http://alizadehlab.stanford.edu/heebo.html), annotations from ReMOAT (PMID 19923232), AILUN (PMID 17971777), AceView (PMID 16925834), and Ensembl (PMID 20459806) databases. Differences in gene expression were considered significant if P < 0.05 after adjustment for multiple testing (Benjamini & Hochberg, J Roy Statist Soc Ser B. 1995;57(1):289-300.), limiting the false discovery rate to <5%. Visualization and plotting was performed using DecisionSite for Functional Genomics (TIBCO Spotfire). Microarray data were deposited in GEO (Accession numbers pending). We performed visualization and plotting using TIBCO Spotfire DecisionSite for Functional Genomics (TIBCO Spotfire, Somerville, MA, USA).
|
| |
|
| Submission date |
Aug 12, 2014 |
| Last update date |
Aug 12, 2014 |
| Contact name |
Anand Champak Patel |
| E-mail(s) |
[email protected]
|
| Organization name |
Washington University School of Medicine
|
| Department |
Pulmonary/Critical Care Medicine
|
| Lab |
Patel
|
| Street address |
Campus Box 8116, 660 S. Euclid Ave.
|
| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL19077 |
| Series (2) |
| GSE60364 |
Mouse Tracheal Epithelial Cell Air-Liquid Interface Primary Culture Time Course (ALI d0, d2, d7) |
| GSE60369 |
Myb permits multilineage airway epithelial cell differentiation |
|
