|
| Status |
Public on Mar 04, 2012 |
| Title |
Cranio072.19April2010 |
| Sample type |
RNA |
| |
|
| Source name |
Human osteoblast cell lines
|
| Organism |
Homo sapiens |
| Characteristics |
age months: 24.23
gender: M
type: Sagittal Synostosis
cell lines: osteoblast
tissue: skull
|
| Biomaterial provider |
The University of Washington (Seattle, WA).
|
| Growth protocol |
Patient osteoblast cell lines were grouped into batches of five. Batches were grouped by age, each containing the following: one control sample, one coronal case, one metopic case, and two cases of sagittal craniosynostosis to reduce bias introduced by culture condition variability. Osteoblast cell lines stored in liquid nitrogen in freezing media consisting of 90% fetal bovine serum and 10% DMSO were thawed prior to culture. All 249 osteoblast lines were grown in T25 flasks containing Waymouth?s Media (Sigma W1625 lot 097K8303) supplemented with 2X antibiotic (100X Pen/Strep/Fungizone, Hyclone SV30079.01, Lot JUA33955) and 10% FBS (Hyclone SH30070.03, Lot ATK33398). Upon reaching 75% confluence, cells were trypsinized using 0.05% Trypsin (Hyclone SH30236.02, Lot J090511), counted and passaged at a cell density of 175,000 cells per 25cm2. All cells were cultured at 37 degrees C, 5% CO2, and 99% humidity.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following the plating of 175,000 cells per 25cm2, each osteoblast cell line was once again grown to 75% confluence, photographed for quality control purposes, washed twice with 1X PBS, and trypsinized. An equal volume of media containing FBS was added after trypsin exposure, and cells were centrifuged twice at 200 x g for 10 minutes at 4degrees C in nuclease free 15ml conical tubes (Corning 430791). In between the centrifugation steps, cells were washed once with 1X PBS. Cell pellets were then kept on ice until RNA extraction. For RNA extraction, Roche High Pure miRNA Isolation Kit was used in accordance with the manufacturer's protocol (Roche 050080576001). RNA was stored immediately in -80 degrees C and submitted for microarray processing on dry ice.
|
| Label |
biotin
|
| Label protocol |
For microarray hybridization, RNA was processed for hybridization to Affymetrix Human Gene 1.0 ST Array through a core facility in the Functional Genomics Laboratory in the Center for Ecogenetics and Environmental Health at the University of Washington according to the manufacturer?s instructions.
|
| |
|
| Hybridization protocol |
The standard hybridization protocol was used as recommended by Affymterix.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000 through the University of Washington Functional Genomics Laboratory in the Center for Ecogenetics and Environmental Health (http://depts.washington.edu/ceeh/ServiceCores/FC1/FC1.html).
|
| Description |
Cranio072.19April2010.CEL
osteoblasts derived from human skull
|
| Data processing |
Affymetrix Expression Console software, RMA normalization log2
|
| |
|
| Submission date |
Mar 15, 2011 |
| Last update date |
Sep 05, 2017 |
| Contact name |
James William MacDonald |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Washington
|
| Department |
Environmental and Occupational Health Sciences
|
| Street address |
4225 Roosevelt Way NE
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98105-6099 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE27976 |
Calvarial osteoblast transcriptome analysis identifies genetic targets and extracellular matrix-mediated focal adhesion as potential biomarkers for single-suture craniosynostosis |
|
