|
| Status |
Public on Aug 30, 2014 |
| Title |
wildtype E10.5 hindlimb buds, biological rep4 |
| Sample type |
RNA |
| |
|
| Source name |
wildtype E10.5 hindlimb buds
|
| Organism |
Mus musculus |
| Characteristics |
genotype: wildtype
tissue: hindlimb buds
|
| Treatment protocol |
Hindlimb limb buds (both right and left) were dissected from E10.5 wildtype and Nipbl+/- embryos
|
| Growth protocol |
Embryos were dissected at day E10.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from hindlimbs of E10.5 mice using Qiagen's RNeasy mini kit with on-column DNA digestion.
|
| 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). Arrays were scanned using the GeneChip Scanner 3000 7G and GeneChip Operating Software v. 1.4 to produce .CEL intensity files
|
| Scan protocol |
Arrays were scanned using the GeneChip Scanner 3000 7G and GeneChip Operating Software v. 1.4 to produce .CEL intensity files
|
| Description |
Control
|
| Data processing |
These probe cell intensity files (.CEL) were analyzed using Expression File Creator by GenePattern (Broad Insitute) using the RMA algorithm, quantile normalization and background correction.
|
| |
|
| Submission date |
Aug 29, 2014 |
| Last update date |
Aug 30, 2014 |
| Contact name |
Arthur D Lander |
| Organization name |
University of California Irvine
|
| Department |
Developmental and Cell Biology
|
| Lab |
Center for Complex Biological Systems
|
| Street address |
2638 Biosciences 3
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92697 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE60932 |
Gene expression changes in limb buds of Nipbl-haploinsufficient mice |
|
