|
| Status |
Public on Jan 31, 2012 |
| Title |
MIBP1 overexpression, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
HEK293 cell clone stably overexpressing MIBP1. Clone No.76
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
|
| Treatment protocol |
HEK293 cells were transfected with pCI-neo (empty vector control) or pCI-neo-FLAG-HA-MIBP1 linearized with AclI and selected at 400 μg/ml G418 (345812, Calbiochem, San Diego, CA) for 2 weeks. Selected cells were cloned using cloning rings and maintained in the presence of G418. Genomic integration and protein expression of MIBP1 were confirmed by genomic PCR and immunoblotting using anti-FLAG M2 antibody (Sigma-Aldrich).
|
| Growth protocol |
HEK293 cells were obtained from Clontech (Mountain View, MO) and maintained in Dulbecco’s modified Eagle’s medium (D5796, Sigma-Aldrich, St. Louis, CA), supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin (15140-122, Invitrogen, Carlsbad, CA), in 5% CO2 at 37 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from triplicated transfectants for each plasmid using RNeasy plus mini kit (74134, Qiagen, Hilden, Germany). RNA quality was checked using 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), and RNA Integrity Number (RIN) was confirmed to be 9.4 or greater for all samples. Total RNA at 200 ng was amplified and reverse-transcribed using GeneChip WT Sense Target Labeling and Control Reagents (900652, Affymetrix).
|
| Label |
Biotin
|
| Label protocol |
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
|
| |
|
| Hybridization protocol |
Samples were hybridized using Affymetrix hybridization kit materials. The cocktails were heated at 99°C for 5 minutes, then 45°C for 5 minutes and centrifuged at max speed for 1 minute. The 80 μl of hyb solution was transfered to each array, then seal the holes with sticker. They were hybridized for 18 hours in 45°C hybridization oven at 60 rpm. Fluidics Script Protocol was FS450_0007
|
| Scan protocol |
Affymetrix GeneChiP Scanner 3000 7G
|
| Description |
FHM-76
|
| Data processing |
Cel files were summarized and normalized with Robust Multi-array Average (RMA) method in Expression Console software version 1.1.1 (Affymetrix). Logarithmic signal intensities were antilog-transformed to linear scale using Expression Console.
probe group file: HuGene-1_0-st-v1.r3.pgf
meta-probeset file: HuGene-1_0-st-v1.r3.mps
|
| |
|
| Submission date |
Jan 04, 2011 |
| Last update date |
Jan 31, 2012 |
| Contact name |
Yuji Iwashita |
| E-mail(s) |
[email protected]
|
| Phone |
+81-92-642-6170
|
| Fax |
+81-92-632-2375
|
| URL |
http://www.gen.kyushu-u.ac.jp/~genome/
|
| Organization name |
Kyushu University, Medical Institute of Bioregulation
|
| Department |
Reseach Center for Genetic Information
|
| Lab |
Division of Genome Analysis
|
| Street address |
3-1-1 Maidashi, Higashi-ku
|
| City |
Fukuoka |
| State/province |
Fukuoka |
| ZIP/Postal code |
812-8582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE26420 |
Expression data from HEK293 cells with or without MIBP1 overexpression |
|
