|
| Status |
Public on Oct 28, 2010 |
| Title |
WT MEF CT30-3 |
| Sample type |
RNA |
| |
|
| Source name |
RNA from WT MEF (30h after serum shock)
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Mouse embryonic fibroblasts
genotype: wild type
time: CT30
|
| Treatment protocol |
Serum shock treated with 50% horse serum (in DMEM) for 2h
|
| Growth protocol |
Cultured in DMEM supplemented with 5% FBS and 5% NCS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was initially isolated using TRIzol Reagent (Gibco BRL Life Technologies, Rockville, MD), and passed through an RNeasy spin column (Qiagen, Chatsworth, CA) for further clean up. Eluted total RNAs were quantified (Nanodrop) with a portion of the recovered total RNA adjust to a final concentration of 100ng/ul.
|
| 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 GenechipÒ 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 GeneChipÒ 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 |
0.54 ug of single-stranded cDNA was hybridized at 45c with rotation for 17 hours (Affymetrix GeneChip® Hybridization Oven 640) to probe sets present on an Affymetrix (GeneChip® 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.
|
| 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 |
Oct 27, 2010 |
| Last update date |
Oct 27, 2010 |
| Contact name |
Sayako Katada |
| E-mail(s) |
[email protected]
|
| Organization name |
UC Irvine
|
| Street address |
Rm2226B GNRB Dep. Pharmacology UC Irvine
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92697 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE24964 |
Expression profiles in WT and MLL1-KO MEF at two different circadian time point |
|
