|
| Status |
Public on Aug 03, 2009 |
| Title |
Monocyte_Ly6Chi_Spleen_01 |
| Sample type |
RNA |
| |
|
| Source name |
Monocyte Ly-6Chi, Spleen
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: female
age: 8-12 weeks
tissue: Spleen
cell type: monocyte
monocyte subset: Ly6Chi
cell signature: Ly-6Chi monocytes were identified as CD11bhi (CD90/B220/CD49b/NK1.1/Ly-6G)lo (F4/80/I-Ab/CD11c)lo
|
| Biomaterial provider |
C57BL/6 mice were purchased from The Jackson Laboratory, Bar Harbor, Maine 04609 USA
|
| Treatment protocol |
healthy mice, no treatment
|
| Growth protocol |
CHOW
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Monocyte subsets from blood and spleen of a group of four mice were isolated by fluorescence activated cell sorting (FACS) as CD11bhi (CD90/B220/CD49b/NK1.1/Ly-6G)lo (F4/80/I-Ab/CD11c)lo Ly-6Chi cells.
Samples of 1,000 Ly-6Chi blood and Ly-6Chi splenic monocytes were collected directly into 20 µl lysis buffer of the PicoPure RNA isolation kit (Arcturus). RNA extraction was subsequently performed according to the manufacturer’s instructions (Arcturus).
Total RNA quality was assessed using RNA pico lab chips on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RIN (RNA integrity number) >= 8.
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified using the NuGen WT-Ovation Pico System, and the amplified cDNA was labeled using the FL-Ovation cDNA Fluorescent Module (NuGen Technologies, San Carlos, Ca). 5ng of input total RNA was reverse–transcribed into cDNA and then amplified using a linear isothermal amplification process (SPIA). The amplified products were CY-3 labeled and fragmented according to manufacturer’s guidelines.
|
| |
|
| Hybridization protocol |
Labeled cDNA was assessed using the Nanodrop ND-100 (Nanodrop Technologies, Inc., Wilmington DE) and the Agilent 2100 Bioanalyzer; equal amounts of Cy3-labeled target were hybridized to Agilent whole mouse genome 4x44K Ink-jet arrays. Hybridizations were performed for 14 h according to the manufacturers protocol.
|
| Scan protocol |
Arrays were scanned using the Agilent microarray scanner. Raw signal intensities were extracted with their Feature Extraction v9.1 software.
|
| Description |
Sample preparation, labeling, and array hybridizations were performed according to standard protocols from the UCSF Shared Microarray Core Facilities and Agilent Technologies (http://www.arrays.ucsf.edu and http://www.agilent.com).
|
| Data processing |
The dataset was normalized using the quantile normalization method. No background subtraction was performed, and the median feature pixel intensity was used as the raw signal before normalization. All procedures were carried out using functions in the R package limma in Bioconductor.
|
| |
|
| Submission date |
Feb 16, 2009 |
| Last update date |
Sep 26, 2011 |
| Contact name |
Martin Etzrodt |
| E-mail(s) |
ETZRODT.MARTIN@MGH.HARVARD.EDU
|
| Phone |
(617) 643-0500
|
| Fax |
(617) 643-6133
|
| URL |
http://csb.mgh.harvard.edu/pittet
|
| Organization name |
Massachusetts General Hospital
|
| Department |
Center for Systems Biology
|
| Lab |
Pittet Lab
|
| Street address |
185 Cambridge Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE14850 |
Microarray analysis of splenic reservoir monocytes and their blood counterparts |
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