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
