|
| Status |
Public on Jul 17, 2012 |
| Title |
Gata2+9.5_090111_wtF |
| Sample type |
RNA |
| |
|
| Source name |
E12.5 murine CD31+ cells, wild type embryo F from litter 2
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6J
litter: 2
genotype/variation: wild type
developmental stage: embryo
age: E12.5
cell type: Magnetic bead-enriched CD31+ cells
|
| Treatment protocol |
CD31+ cells were enriched from individual embryo carcasses lacking fetal livers and brains. Single cell suspensions were prepared by collagenase treatment and mechanical dissociation. CD31+ cells were isolated with anti-CD31 antibody-bound magnetic beads.
|
| Growth protocol |
Gata2 +9.5 mutant and wild type E12.5 littermates were obtained from matings of Gata2 +9.5+/- males and females.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from CD31+ cells using Trizol reagent (Invitrogen) and futher purified using Qiagen microRNA columns following manufacturer's recommendations. RNA was quantified using NanoDrop-1000 and and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent. On completion of the fragmentation reaction, 25 ul of 2x GE hybridization buffer (HI-RPM) was added to the fragmentation mixture and hybridized to Agilent Sureprint G3 Mouse GE 8x60 Microarray (GPL13912), microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), both containing 0.005% Triton X-102Slides were air dried.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using the 8x60K G3 one color Microarray format. Dye channel is green, scan area 61x21.65 mm, scan resolution 3 um, Tiff is 20 bit.
|
| Description |
Gene expression in CD31+-enriched cells from wild type E12.5 embyo
Wild Type sample 4
|
| Data processing |
We median centered the arrays of four independent wild type and +9.5-/- embryos and filtered out the bottom 10% of the probes with the lowest standard deviation across combined wild-type and mutant samples. The differential expression analysis between the mutant and the wild-type samples was conducted with the R package Limma
|
| |
|
| Submission date |
Jul 12, 2012 |
| Last update date |
Jul 17, 2012 |
| Contact name |
Kirby Dean Johnson |
| E-mail(s) |
kjohns25@wisc.edu
|
| Organization name |
University of Wisconsin-Madison
|
| Department |
Pharmacology
|
| Lab |
Emery H. Bresnick
|
| Street address |
1300 University Avenue
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE39300 |
Gene expression changes in embryonic PECAM-1+ cells from Gata2 +9.5 enhancer targeted mice |
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