|
| Status |
Public on Jul 17, 2009 |
| Title |
Expression profiling of Bmal -/- littermate dorsal skin_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Bmal -/- dorsal skin at P22, biological replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
Age: postnatal day 22
Gender: female
Tissue: dorsal skin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Biotin
|
| Label protocol |
Single-stranded, then double-stranded cDNA was synthesized from the poly(A)+mRNA present in the isolated total RNA (typically 100ng total RNA starting material each sample reaction) using the GeneChip WT cDNA synthesis Kit (Affymetrix, Inc., Santa Clara, CA) and random hexamers tagged with a T7 promoter sequence. The double-stranded cDNA is then used as a template to generate many copies of antisense cRNA from an in vitro transcription reaction (IVT) of 16hrs in the presence of T7 RNA Polymerase using the Affymetrix Genechip WT cDNA Amplification Kit. 10 ug of cRNA were input into the second cycle cDNA reaction with random hexamers that are used to reverse transcribe the cRNA from the first cycle to produce single-stranded DNA in the sense orientation.
The single-stranded DNA sample is fragmented (WT Terminal Labeling Kit, Affymetrix, Inc, Santa Clara, CA) to an average strand length of 60 bases (range 40-70bp) following prescribed protocols (Affymetrix GeneChip WT Sense Target Labeling Assay Manual). The fragmented single-stranded DNA is subsequently labeled with recombinant terminal deoxynucleotidyl transferase (TdT) and the Affymetrix proprietary DNA Labeling Reagent that is covalently linked to biotin.
|
| |
|
| Hybridization protocol |
Following the recommended procedure, 0.54 ug of this fragmented target single-stranded cDNA was hybridized at 45c with rotation for 17 hours (Affymetrix GeneChip Hybridization Oven 640) to probe sets present on an Affymetrix Mouse Gene 1.0 ST array. The GeneChip arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fludics Station 450 (Fluidics protocol FS450_007).
|
| Scan protocol |
Arrays were scanned using the GeneChip Scanner 3000 7G and GeneChip Operating Software v. 1.4 to produce .CEL intensity files.
|
| Description |
Hair cycle staging: telogen
|
| Data processing |
These probe cell intensity files (*.CEL) were analyzed in Affymetrix Expression Console software v1.1 using the PLIER algorithm to generate probe level summarization files (*.CHP).
(Algorithm: PLIER v 2.0; Quantification Scale: Linear; Quantification Type: Signal and Detection P-Value; Background: PM-GCBG; Normalization Method: Sketch-Quantile).
|
| |
|
| Submission date |
Dec 16, 2008 |
| Last update date |
Jul 17, 2009 |
| Contact name |
Kevin K Lin |
| E-mail(s) |
[email protected]
|
| Organization name |
University of California, Irvine
|
| Department |
Biological Chemistry & Medicine
|
| Lab |
Bogi Andersen
|
| Street address |
250 Sprague Hall
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92697-4030 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE14006 |
Expression profiling of Bmal mutant dorsal skin at telogen of hair follicle cycling |
|
