Comment[ArrayExpressAccession]	E-MTAB-2334
MAGE-TAB Version	1.1
Investigation Title	Expression profile of oesophageal epithelium basal cells comparing DNMaml expressing cells versus controls
Comment[Submitted Name]	Expression profile of oesophageal epithelium basal cells comparing DNMaml expressing cells versus controls
Experiment Description	Multiple cancers may arise from within a clonal region of preneoplastic epithelium, a phenomenon termed M-^Qfield changeM-^R. However, it is not known how field change develops.  We investigate this question and analyse the behavior of scattered single esophageal epithelial progenitor cells expressing a mutation that inhibits the Notch signaling pathway (Dominant negative Maml).
Experimental Design	development or differentiation design	in_vivo_design	co-expression_design
Comment[AEExperimentType]	transcription profiling by array
Comment[AEExperimentDisplayName]	Transcription profiling by array of mouse oesophageal epithelium basal cells comparing DNMaml expressing cells versus controls
Experimental Factor Name	genotype
Experimental Factor Type	genotype
Quality Control Type	biological replicate
Quality Control Term Source REF	EFO
Public Release Date	2014-04-08
Person Last Name	Fowler
Person First Name	Jo
Person Mid Initials
Person Email	jf384@hutchison-mrc.cam.ac.uk
Person Phone
Person Address	MRC-Cancer Unit, University of Cambridge, Hutchison/MRC research centre, Box 197 Biomedical campus, Cambridge CB2 0XZ
Person Affiliation
Person Roles	submitter
PubMed ID
Publication Author List	Maria P. Alcolea, Philip Greulich, Agnieszka Wabik, Benjamin D. Simons,Philip H. Jones
Publication Title	Differentiation imbalance in single esophageal progenitor cells causes clonal immortalization and field change
Publication Status
Protocol Name	P-MTAB-37695	P-MTAB-37696	P-MTAB-37697	P-MTAB-37698	P-MTAB-37699	P-MTAB-37700	P-MTAB-37701	P-MTAB-37702
Protocol Type	growth protocol	treatment protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning protocol	normalization data transformation protocol	normalization data transformation protocol
Protocol Description	Experimental mice were doubly heterozygous for the inducible cre allele AhcreERT and a conditional allele encoding a dominant negative mutant of mastermind-like1 (DNM) fused to Green Fluorescent Protein (GFP),  downstream of a LoxP flanked M-^QStopM-^R cassette targeted to the Rosa 26 locus (AhcreERTR26flDNM-GFP/wt, DNM)14. In these mice, transcription of a cre mutant estrogen receptor fusion protein (creERT) is induced by napthoflavone. Tamoxifen is also required for creERT to gain access to the nucleus.  In the presence of both drugs, creERT removes the M-^SStopM-^T cassette resulting in DNM expression in the cell and its progeny.  Controls included both age-matched un-induced DNM mice as well as the previously characterized AhcreERTR26flEYFP/wt (YFP) strain, which expresses Yellow Fluorescent Protein (YFP) from the Rosa 26 locus following cre induction. 	Low frequency expression of DNMaml in the mouse esophagus was achieved by inducing animals aged 8-12 weeks with an intraperitoneal dose of 80ug/kg Napthoflavone and 0.3mg Tamoxifen. Two cohorts of 3 pooled animals was used to isolate basal OE cells by Facs 15 days post induction.	Epithelial sheets were minced and single-cell suspension obtained using gentleMACS Dissociator (Miltenyi Biotec) followed by filtration through a 30um cell strainer. Cells were centrifuged and resuspended in 1% Fetal Bovine Serum in PBS and stained for the basal cell marker a6 integrin (BioLegend; 313610; 5ug/ml) for 30 minutes at 4C. An isotype control was used to exclude unspecific immunoglobulin binding (BioLegend; 400526; 5ug/ml). Endogenous GFP levels allowed the identification of DNMaml recombinant cells. 7AAD (2ug/ml) was used to determine cell viability. Two populations of viable single cells were sorted (GFP+/a6+ and GFP-/a6+) on a MoFlow cell sorter (Dako Cytomation) and collected for further RNA analysis.  The quality of the RNA was determined by using an Agilent 2100 Bioanalyzer. Samples used for hybridization had a RNA Integrity Number = 7.8. Due to the limited amount of starting material, and therefore, the low RNA yield, the RNA was amplified using a NuGen, Ovation Pico WTA 3300-60 kit according manufacturer's instructions.	200ng of the amplified RNA was labelled using the  Illumina TotalPrep -96 RNA Amplification kit	RNA was hybridised to Mouse Whole Genome-6 v2 Illumina array following Illumina protocols	Arrays were scanned using the Illumina bead array reader	The resulting data were transformed using the Variance Stabilization Transformation (VST) from lumi package, to avoid the bias at low and high intensities, and normalized by quantile normalization. The paired comparisons between GFP+ and GFP- were then performed using the limma package (Linear Models for Microarray Data, Bioconductor) and the results corrected for multiple testing using False Discovery Rate (FDR)	Paired compairson between GFP-plus and GFP minus arrays using limma package (Linear Models for Microarray Data, Bioconductor) and corrected for FDR
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO	EFO	EFO
Term Source Name	EFO
Term Source File	http://www.ebi.ac.uk/efo
SDRF File	E-MTAB-2334.sdrf.txt