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Sample GSM431672 Query DataSets for GSM431672
Status Public on Jul 31, 2009
Title Fibroblasts_individual_UC49
Sample type RNA
 
Source name Primary fibroblasts
Organism Homo sapiens
Characteristics cell type: Primary fibroblasts
gender: Male
Treatment protocol Cord blood was collected in 50 ml falcon tubes containing 10 ml of anti-coagulants (Sodium citrate, EDTA- Sigma, St. Louis, MO) and kept at 4oC for less than 24 hours prior to treatment. For separation, cord blood was diluted 2 fold in PBS (Invitrogen), layered on Ficoll-Paque (GE Healthcare Lifesciences, Chalfont St. Giles, United Kingdom) and centrifuged for 30 min at 800g. The mononuclear cell layer was removed, washed twice in 40 ml of PBS and re-suspended in 1 ml of RPMI 20% FCS, 1% antibiotics (Amimed, Basel, Switzerland). For fibroblast preparation, cord tissue was finely cut under sterile conditions in 1 ml DMEM 10% FCS, 1% antibiotics (Amimed), transferred to a T25 flask and cultured upside-down for 12 hours to allow cells to attach to plastic. Flasks were then turned and left for about 1 week until fibroblast clusters appear. Fibroblasts were then expanded with standard procedures. For preparation of EBV-immortalized lymphoblastoid cell lines (LCLs), 300 ul of re-suspended cells and 100 ul of EBV were transferred to a 24-well plate well, and cultured in an incubator at 37 oC, 5 % CO2. Fresh medium was added and replaced every 2-3 days. Cells were kept in culture for no less than 21 days prior to freezing. For PHA stimulated T-cell preparation, re-suspended mononuclear cells were diluted to a concentration of 1 x 106 cells/ml in RPMI (Invitrogen) with 5 ug/ml of PHA (Sigma), and cultured for 5 days with 2/3 medium replacement after 2.5 days. A subset of samples was characterized by flow cytometric analysis for expression of CD3, CD25 and CD69 (Becton Dickinson, Franklin Lakes, NJ) revealing a homogenous activated T-cell population.
Growth protocol Cord blood was collected in 50 ml falcon tubes containing 10 ml of anti-coagulants (Sodium citrate, EDTA- Sigma, St. Louis, MO) and kept at 4oC for less than 24 hours prior to treatment. For separation, cord blood was diluted 2 fold in PBS (Invitrogen), layered on Ficoll-Paque (GE Healthcare Lifesciences, Chalfont St. Giles, United Kingdom) and centrifuged for 30 min at 800g. The mononuclear cell layer was removed, washed twice in 40 ml of PBS and re-suspended in 1 ml of RPMI 20% FCS, 1% antibiotics (Amimed, Basel, Switzerland). For fibroblast preparation, cord tissue was finely cut under sterile conditions in 1 ml DMEM 10% FCS, 1% antibiotics (Amimed), transferred to a T25 flask and cultured upside-down for 12 hours to allow cells to attach to plastic. Flasks were then turned and left for about 1 week until fibroblast clusters appear. Fibroblasts were then expanded with standard procedures. For preparation of EBV-immortalized lymphoblastoid cell lines (LCLs), 300 ul of re-suspended cells and 100 ul of EBV were transferred to a 24-well plate well, and cultured in an incubator at 37 oC, 5 % CO2. Fresh medium was added and replaced every 2-3 days. Cells were kept in culture for no less than 21 days prior to freezing. For PHA stimulated T-cell preparation, re-suspended mononuclear cells were diluted to a concentration of 1 x 106 cells/ml in RPMI (Invitrogen) with 5 ug/ml of PHA (Sigma), and cultured for 5 days with 2/3 medium replacement after 2.5 days. A subset of samples was characterized by flow cytometric analysis for expression of CD3, CD25 and CD69 (Becton Dickinson, Franklin Lakes, NJ) revealing a homogenous activated T-cell population.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from fibroblasts, LCLs, and T-cells and was prepared with RNeasy columns with on-column DNAse treatment (Qiagen, Venlo, The Netherlands), quantified with NanoDrop (Thermo Scientific, Waltham, MA) and analyzed with a 2100 Bioanalyzer (Agilent, Santa Clara, CA). Two one-quarter scale Message Amp II reactions (Ambion, Foster City, CA) were performed for each RNA extraction with 200 ng of total RNA.
Label biotin
Label protocol Biotinylated cRNA was prepared through two one-quarter scale Message Amp II reactions (Ambion, Foster City, CA).
 
Hybridization protocol Standard Illumina hybridization protocol
Scan protocol Standard Illumina scanning protocol
Description One of three cell types derived from umbilical cords of 85 individuals
Data processing Intensity values were log2 transformed and normalized independently for each cell type (quantile normalization for sample replicates, and median normalization across individuals). Each cell type was then renormalized using the mean of the medians of each cell type expression values
 
Submission date Jul 21, 2009
Last update date Jul 21, 2009
Contact name Emmanouil T Dermitzakis
E-mail(s) emmanouil.dermitzakis@unige.ch
Phone +41 (0) 22 379 5483
Organization name University of Geneva Medical School
Department Department of Genetic Medicine and Development
Lab Population and comparative genomics
Street address 1 Rue Michel-Servet
City Geneva
ZIP/Postal code 1211
Country Switzerland
 
Platform ID GPL6884
Series (1)
GSE17080 Common regulatory variation impacts gene expression in a cell type dependent manner

Data table header descriptions
ID_REF
VALUE Log2 transformed normalized intensities

Data table
ID_REF VALUE
ILMN_1809034 8.848489623
ILMN_1660305 9.511074066
ILMN_1792173 8.973525108
ILMN_1762337 6.705083504
ILMN_2055271 7.021613461
ILMN_1736007 6.733226088
ILMN_1814316 6.699302403
ILMN_2359168 6.631899953
ILMN_1731507 6.552804705
ILMN_2136495 6.396459404
ILMN_1735045 9.20235901
ILMN_1680754 6.889012141
ILMN_1755321 7.576227171
ILMN_1698554 9.354349666
ILMN_1760414 6.796795036
ILMN_2061446 10.57297143
ILMN_1676336 10.46062138
ILMN_1752884 6.695564447
ILMN_2270015 7.096495155
ILMN_1809959 6.463504211

Total number of rows: 22651

Table truncated, full table size 550 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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