|
| Status |
Public on Sep 06, 2013 |
| Title |
1h_shh_replicate_2_chip_A |
| Sample type |
RNA |
| |
|
| Source name |
PN4-5 cerebellar culture_1h_shh
|
| Organism |
Mus musculus |
| Characteristics |
strain: Swiss-Webster B
age: postnatal day 4-5 (PN4-5)
cell type: primary cerebellar culture (pooled from litter mates)
treated with: shh for 1h
microarray hybridization date: 2000-10-18
|
| Treatment protocol |
Described in PMID:11960025
|
| Growth protocol |
Described in PMID:11960025
|
| Extracted molecule |
total RNA |
| Extraction protocol |
20 micrograms of total RNA was used to generate target cRNA for hybridization to Affymetrix Mu11K chipset subA and subB oligonucleotide microarrays. A single chip set was used for each time-point. Before target generation, individual RNA samples were pooled so that each target was derived from a minimum of 3 individual cerebella. This step was performed to minimize both biologic variability and the number of microarrays necessary to generate informative data. After pooling, RNA was re-purified using Qiagen purification kits (Valencia, CA). Target cRNA was synthesized according to manufacturer's protocol. Briefly, total RNA was reverse transcribed into a complete cDNA library (SuperScript Choice kit; Invitrogen) using a T7/T24 primer (Genset Corp., La Jolla, CA). This cDNA was subsequently in vitro transcribed into biotin-labeled cRNA (High Yield RNA Transcript Labeling kit; Enzo Diagnostics, Farmingdale, NY). The resulting cRNA was fragmented and assessed for quality by gel electrophoresis. Under standard conditions processing of RNAs for GeneChip Analysis was in accordance with methods described in the Affymetrix GeneChip Expression Analysis Technical Manual. Synthesis of cDNA first and second strand is performed using the GeneChip Expression 3’-Amplification Reagents One-Cycle cDNA Synthesis Kit (P/N 900431). Cleanup of the double stranded product is carried according to standard Affymetrix protocols.
|
| Label |
Biotin
|
| Label protocol |
In vitro transcription (IVT) is performed using the GeneChip Expression Amplification Reagents kit- 30 reactions (P/N 900449) and is carried out according to the standard Affymetrix protocols and quantification of the IVT samples is carried out on a Bio-Tek UV Plate Reader.
|
| |
|
| Hybridization protocol |
25 micrograms of each biotin-labeled cRNA was hybridized to an Affymetrix Mu11K sub A and B chip according to the manufacturer's protocol. Four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) were included in each hybridization reaction to verify consistent hybridization efficiency. Hybridization is carried out according the Affymetrix GeneChip® Manual. Affymetrix hybridization ovens are used to incubate the arrays at a constant temperature of 45oC overnight. Preparation of microarrays for scanning is carried out with Affymetrix appropriate wash protocols matched to the specific chip type on a Model 450 Fluidics station. Affymetrix GeneChip Operating Software (GCOS) operating system controls the Fluidics station process.
|
| Scan protocol |
Standard Affymetrix protocol.
|
| Description |
1h_shh_2_chipA__CL2000101811AA
PN4-5 cerebellar culture, 1h, shh, chip_A
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.
|
| |
|
| Submission date |
Sep 05, 2013 |
| Last update date |
Sep 06, 2013 |
| Contact name |
Alvin T. Kho |
| E-mail(s) |
alvin_kho@hms.harvard.edu
|
| Phone |
617-919-2182
|
| Organization name |
Boston Children's Hospital
|
| Department |
Informatics Program
|
| Street address |
320 Longwood Avenue
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL75 |
| Series (1) |
| GSE50606 |
Identification of genes expressed with temporal-spatial restriction to developing cerebellar neuron precursors by a functional genomic approach perspective of human cancers. |
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