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Sample GSM538745 Query DataSets for GSM538745
Status Public on Jan 12, 2011
Title CD34+CD200+CD49+Replicate4
Sample type RNA
 
Source name CD34+CD200+CD49+
Organism Mus musculus
Characteristics tissue: Back Skin
sex: female
age: 50 days
hair stage: telogen
Treatment protocol Cells were not treated
Growth protocol Cells were not expanded but isolated enzymatically from mouse skin
Extracted molecule total RNA
Extraction protocol RNAeasy Qiagen protocol
Label biotin
Label protocol Briefly, 5ug of total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP.
 
Hybridization protocol The cRNA products were fragmented to 200 nucleotides or less, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to U133A Affymetrix microarray for human and 430 v2 for mouse. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
Scan protocol A confocal scanner was used to collect fluorescence signal at 3um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature.
Description Gene expression data from FACS sorted mouse adult keratinocytes using cell surface markers
Data processing Affymetrix GeneChip Operating System (GCOS, v. 1.4, Affymetrix, Inc.) was used to quantitate expression signal levels for the arrays; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. These values were exported as .cel files. The average of the lowest 2% of probe intensities occurring in each of 16 microarray sectors was set as background and subtracted from all features in that sector. Probe pairs were scored positive or negative for detection of the targeted sequence by comparing signals from the perfect match and mismatch probe features. The number of probe pairs meeting the default discrimination threshold (tau = 0.015) was used to assign a call of absent, present or marginal for each assayed gene, and a p-value was calculated to reflect confidence in the detection call. The flag values were additionally exported in .chp files.
Affymetrix probe intensities were imported into GeneSpring (v7.2, Agilent Technologies) where probeset signal values were calculated using the GC-RMA algorithm. Upon import of the Affymetrix flag values, the probesets were filtered to retain only those that were flagged P (present) in at least two of the repeat samples. This list was used for condition-based principle components analysis (PCA) to assess global trends in sample similarity. This analysis demonstrated groupings based on sample identity and prompted the use of mixed-model 2-way ANOVA as a means of finding differentially regulated genes between the sites of interest.
GC-RMA signal data was imported into Partek Genomics Solution (PGS, v6.2, Partek, Inc.), where the data were log2 transformed. Probesets not shared between mouse and human chips were removed. Genes less than p<0.05 in p-values for the identity of sample term of the ANOVA, and less than 2 fold changes between positive and negative populations were also excluded. The resulting enriched gene lists for bulge, secondary hair germ, and CD200highITGA6high cells were then examined for intersection using Partek.
 
Submission date Apr 28, 2010
Last update date Jan 12, 2011
Contact name Luis Andres Garza
E-mail(s) LAG@jhmi.edu
Phone 410-955-8662
Organization name Johns Hopkins School of Medicine
Department Dermatology
Lab Garza lab
Street address 1550 Orleans St. Suite 204 CRBII Koch
City Baltimore
State/province MD
ZIP/Postal code 21287
Country USA
 
Platform ID GPL1261
Series (2)
GSE21568 Mouse bulge (CD34+CD200+CD49+) versus secondary hair germ (CD34-CD200+CD49+) versus interfollicular epidermis (CD34-CD200-CD49+)
GSE21569 Bald scalp retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells

Data table header descriptions
ID_REF
VALUE GC-RMA signal (GeneSpring)

Data table
ID_REF VALUE
1424549_at 5.766550064
1449833_at 5.548970222
1419555_at 5.242919922
1418735_at 7.116839886
1435792_at 7.453989983
1436279_at 3.409800053
1436869_at 3.131930113
1457777_at 6.055900097
1426147_s_at 4.629879951
1425122_at 4.708159924
1448752_at 7.127970219
1438394_x_at 7.223060131
1422240_s_at 8.008119583
1448982_at 6.738510132
1416980_at 3.565700054
1457867_at 3.040960073
1437360_at 3.708790064
1423476_at 6.153049946
1446631_at 6.127910137
1416287_at 6.93900013

Total number of rows: 45101

Table truncated, full table size 1026 Kbytes.




Supplementary file Size Download File type/resource
GSM538745.CEL.gz 3.7 Mb (ftp)(http) CEL
GSM538745.CHP.gz 387.9 Kb (ftp)(http) CHP
Processed data included within Sample table
Processed data provided as supplementary file

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