|
| Status |
Public on Apr 08, 2011 |
| Title |
GCB LRF F/+ mb1-Cre+ #2 |
| Sample type |
RNA |
| |
|
| Source name |
Germinal center B cells of control mouse
|
| Organism |
Mus musculus |
| Characteristics |
cell type: FACS-sorted germinal center B cells (B220+CD19+CD38-Fas+)
genetic background: mixed including C57BL/6 and Zbtb7a-neo/+N1
genotype: LRF Flox/+ mb1-Cre+ (LRF heterozygous)
age: 10 wk
|
| Treatment protocol |
LRF knockout (LRF Flox/Flox mb-1 Cre+) and control (LRF Flox/+ mb-1 Cre+) mice (4 each) were immunized with sheep red blood cells (SRBCs), and GCB cells were FACS-sorted 7 d later.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA samples were prepared using the Absolutely RNA Microprep kit (Stratagene).
|
| Label |
biotin
|
| Label protocol |
Synthesis and labelling of cDNA targets, hybridization and scanning of GeneChips were carried out by the Microarray Core Facility at City of Hope. Due to limited starting material, we used a modified two-cycle amplification protocol. Briefly, cRNA was generated using 10 ng total RNA, according to the manufacturer's protocol, by using Affymetrix's GeneChip Whole Transcript Sense Target Labelling Assay in the first cycle of amplification. The cRNA was subjected to the RiboMinus kit (Invitrogen) to remove rRNA.
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| |
|
| Hybridization protocol |
The resulting cRNA was used as the template for another round of amplification using the sense target labelling assay kit. Hybridization cocktails containing 5.5 μg of fragmented, end-labelled cDNA were prepared and applied to GeneChip Mouse Gene 1.0 ST arrays (GPL6246, Affymetrix). Hybridization was performed for 16 h, and the arrays were washed and stained with the GeneChip Fluidics Station 450 using FS450_0007 script
|
| Scan protocol |
Arrays were scanned at 5 μm resolution using the Affymetrix GCS 3000 7G and GeneChip Operating Software v. 1.4 to generate CEL intensity files.
|
| Description |
10 wks old mice. 7 days after immunization.
Gene expression data from FACS-sorted germinal center B cells (B220+CD19+CD38-Fas+)
|
| Data processing |
The raw data is normalized by Robust Multi-array Average(RMA) using Affymetrix Expression Console
|
| |
|
| Submission date |
Apr 07, 2011 |
| Last update date |
Apr 08, 2011 |
| Contact name |
Takahiro Maeda |
| Organization name |
Beckman Research Institute of the City of Hope
|
| Department |
Hematopoietic Stem Cell and Leukemia Research
|
| Lab |
Maeda
|
| Street address |
1500E Duarte Rd
|
| City |
Duarte |
| State/province |
CA |
| ZIP/Postal code |
91010 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE28449 |
Expression data from control and LRF-deficient mouse germinal center B cells |
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