|
| Status |
Public on Oct 30, 2012 |
| Title |
Father_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
normal forearm skin biopsy
|
| Organism |
Homo sapiens |
| Characteristics |
disease status: normal
tissue: forearm skin biopsy
cell type: fibroblasts
gender: male
cell line: HGFDFN168
passage: 16
|
| Growth protocol |
Cells were grown under 5% CO2 in MEM supplied by 15% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from 1 million cells with the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. Total RNAs were prepared according to Affymetrix protocols (Affymetrix, Inc). RNA quality and quantity were ensured using the Bioanalyzer (Agilent, Inc.) and NanoDrop (Thermo Scientific, Inc.), respectively.
|
| Label |
biotin
|
| Label protocol |
For RNA labeling, 1 microgram of total RNA was used in conjunction with the Affymetrix recommended protocol for the GeneChip® WT Sense Target Labeling. The total RNA was first subjected to a ribosomal RNA reduction using the Invitrogen RiboMinus Transcriptome Isolation Kit. The remaining RNA was then reverse transcribed in the presence of dT7N6 primers to generate cDNA. An in vitro transcription was then performed and the resulting cRNA was purified. A first strand cDNA was generated from the cRNA in the presence of dUTPs. After RNA hydrolysis, the single-stranded cDNA was fragmented and end labeled with biotin.
|
| |
|
| Hybridization protocol |
The hybridization cocktail containing the fragmented and biotin-labeled cDNAs were hybridized to an Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols as described by Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Molecular Probes, Carlsbad, CA) and enhanced by using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA).
|
| Scan protocol |
An Affymetrix GeneChip Scanner 3000 was used to scan the probe arrays. Gene expression intensities were calculated using the Affymetrix Expression Console software and the CEL files generated.
|
| Description |
KC_10-1 patient168
Patient's healthy father.
|
| Data processing |
CEL files were processed with RMAExpress using Background Adjustment, Quantile Normalization, and Median Polish.
|
| |
|
| Submission date |
Oct 22, 2012 |
| Last update date |
Nov 18, 2016 |
| Contact name |
Rachel Patton McCord |
| E-mail(s) |
Rachel.McCord@umassmed.edu
|
| Phone |
508-856-4377
|
| Organization name |
University of Massachusetts Medical School
|
| Department |
Program in Gene Function and Expression
|
| Lab |
Job Dekker Lab
|
| Street address |
364 Plantation St. LRB 570M
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (2) |
| GSE41751 |
Correlated alterations in genome organization, histone methylation, and DNA-lamina interactions in Hutchinson-Gilford progeria syndrome (expression) |
| GSE41764 |
Correlated alterations in genome organization, histone methylation, and DNA-lamina interactions in Hutchinson-Gilford progeria syndrome |
|
