|
| Status |
Public on Mar 29, 2012 |
| Title |
Macrophage_150k Pa_18 hrs_replicate b |
| Sample type |
RNA |
| |
|
| Source name |
Macrophage_150k Pa_18 hrs
|
| Organism |
Mus musculus |
| Characteristics |
cell type: RAW 267.4 macrophages
matrix stiffness: 150K Pascals
duration: 18 hrs
|
| Treatment protocol |
RAW 264.7 mouse macrophage cells were cultured in complete DMEM medium(10% heat-inactivated FCS), lifted off with trypsin-EDTA (GIBCO) and transferred to polyacrylamide gels for 24 hours before use. Polyacrylamide gel substrates were prepared according to previously described protocol(Krishnan et al., 2009). Concentrations for 1.2 kPa:5% Acrylamide and 0.03% Bis-acrylamide and for 150 kPa:20% Acrylamide and 0.2% Bis-acrylamide. Following gelation, the surface was activated with 200ul of a solution containing 1mM Sulfosuccinimidyl-6-[4-azido-2-nitrophenylamino] hexanoate (Sulfo-SANPAH; Pierce, Rockford, IL), coated with poly-L-lysine. Cells were allowed to adhere overnight on gels before performance of downstream experiments.
|
| Growth protocol |
Cells were cultured on polyacrylamide gels of specific stiffnesses that were coated with poly-L-lysine to facilitate cellular attachment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each experimental sample, RNA quality was assessed by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. Concentrations may also be determined using a NanoDrop 1000 spectrophotometer. Under standard conditions processing of RNAs for GeneChip Analysis was in accordance with methods described in the Affymetrix GeneChip Expression Analysis Technical Manual, revision four, as subsequently detailed. 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 using the Affymetrix GeneChip Cleanup Module (Affymetrix Catalog # 900371).
|
| 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 |
Hybridization is carried out according the Affymetrix GeneChip® Manual. Twenty micrograms of IVT material is the nominal amount used on the GeneChip® arrays. Affymetrix hybridization ovens are used to incubate the arrays at a constant temperature of 45C overnight.
|
| Scan protocol |
Scanning is carried out on a Affymetrix GeneChip® Scanner 3000 7G scanner with autoloader. The Affymetrix GCOS v1.3 operating system controls the Model 3000 7G scanner and data acquisition functions. GCOS maintains the mediated first level data analysis and desktop data management for the entire GeneChip System.
|
| Description |
microarray hybridization data 06/14/2010
Koziel_20.CEL
|
| Data processing |
All 21 sample CEL files were input into Bioconductor/R (version 2.11.10) to derive Robust Multi-Array Average (RMA) normalized output using the rma function in the affy library.
|
| |
|
| Submission date |
Mar 28, 2012 |
| Last update date |
Mar 29, 2012 |
| 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 |
GPL11180 |
| Series (1) |
| GSE36878 |
Cell elasticity determines macrophage function. |
|
