Expression Database

GUDMAP:12317

Theiler Stage

Images

Supplemental Data Files

CEL file:	CRYM_P1_3.CEL
CHP file:	CRYM_P1_3_rma_gene_default.chp
RPT file:	NA
TXT file:	CRYM_P1_3.TXT

Archive ID

160

Principal Investigator(s)

Steve Potter, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA, steve.potter@cchmc.org

Submitted By

Eric Brunskill, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA, brune0@cchmc.org

Specimen Details

Sample GEO ID:

GSM429025

Sample Description:

P1 cap mesenchyme

Title:

CRYM_P1_3

Component(s) Sampled:

cap mesenchyme (EMAP:30284)

Organism:

Mus musculus

Strain:

CD-1

Genotype:

Non-wild type Allele:
Gene		Crym
MGI ID		MGI:4846969
MGI Symbol or lab name		Tg(Crym-EGFP)82Gsat
Type		transgenic insertion
Notes:		Crym BAC-transgenic mice were received from the GENSAT Project (Gene Expression Nervous System Atlas), a NIH-funded consortium that generates transgenic BAC-EGFP reporter lines.

Sex:

Unknown

Development Age:

Theiler Stage:

Pool Size:

approx 4 P1 kidneys

Pooled Sample:

Yes

Dissection Method:

Trypsinization and fluorescent activated cell sorting (FACS).

Experimental Design:

Crym transgenic mice were time-mated. Pregnant Crym transgenic mice were euthanized by standard carbon dioxide asphyxiation. Neonatal pups were fostered to new CD-1 females. At the appropriate time, pups were killed by decapitation with a scalpel and the kidneys were isolated and placed in ice-cold PBS. The kidneys were minced with a razor blade and incubated in the presence of 300µl of 0.25% trypsin-EDTA for 5 minutes at 37ºC. After incubation, the kidneys were further dissociated by titurating in the presence of 600µl of ice-cold 10%FBS/PBS, and then pelleted at 5000 rpm, 4ºC for 5 minutes. The cells were resuspended in 200µl of ice-cold 2%FBS/PBS, filtered using a 70 micron mesh and isolated using FACS.

Array Hybridization

Extracted Molecule:	Total RNA
A260:280 Ratio:	NA
RNA Extraction Protocol:	Potter protocol: 'RNA purification'
Target Amplification Manufacturer/kit:	WT-Ovation Pico RNA Amplification System (NuGEN)
Target Amplification Protocol:	Potter protocols
Rounds of Amplification:	1
Amount Labeled Target Hybridized To Array:	2.5 µg
Label:	Biotin
Label Protocol:	The FL-Ovation cDNA Biotin Module V2 (NuGEN)
Array Hyb/Wash Protocol:	Affymetrix Standard Protocol. The arrays were washed and stained using a Fluidics Station 450 (Affymetrix) utilising the fluidics protocol FS450-0007.
Scan Protocol:	Probe arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G and Affymetrix scanning software Genechip Operating Software Version 1.4.
GCOS Tgt Value:	NA
Data Analysis Method:	Affymetrix Expression Console and GeneSpring 10
Reference:	PND 0.5 whole embryo RNA

Series Details

Series GEO ID:	GSE17139
Number of Samples:	15 samples
Title:	Gene expression profiles of cap mesenchyme and renal vesicle isolated between P0-P4 from Crym-EGFP neonatal transgenic mice using FACS. (GUDMAP Series ID: 28)
Summary:	The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing kidney. The central thesis is straightforward. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing kidney. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in FACS isolated components of the developing kidney. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone.
Type:	Expression profiling of kidney components by microarray
Overall Design:	At E18.5, pups were isolated from CRYM-BAC transgenic mice and fostered to CD-1 mice. Cap mesenchyme cells were isolated from the kidney by fluorescent activated cell sorting (FACS) on postnatal days 0, 1, 2, 3, and 4. RNA was isolated from FACS sorted cells and the gene expression profiles were determined by microarrays.

Platform Details

Platform GEO ID:	GPL6246
Title:	Affymetrix Mouse Gene 1.0 ST Array
Distribution:	commercial
Technology:	in situ oligonucleotide
Organism:	Mus musculus
Manufacturer:	Affymetrix
Manufacturer Protocol:	N/A
Catalogue Number:	N/A