We cultured N=30 Coriell Cell Repository lymphoblastoid cell lines, in triplicate under standardized conditions; when cells per milliliter exceeded 2X107, the cells were harvested, the pellets were washed once with PBS and frozen at -80ºC. Same lot cell culture reagents were used, with three technicians each dedicated to culturing N=10 of these cell lines, with replicate cell cultures cultured in series.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from frozen cell pellets using the Qiagen RNeasy Midi-kit (Valencia, CA). Ten cell culture pellets were extracted at a time by a single technician with a single assistant; cell pellets were removed one-at-a-time from the -80ºC freezer, quick thawed by rubbing between gloved hands, and Qiagen denaturant immediately added. Ethanol was added to each sample, vortexed, and the samples applied to Qiagen Midi columns, washed as specified, treated with RNase-free DNase “on-column”, followed by additional washes before elution of the RNA with the provided buffer. After elution, sample volume was determined by weight, sodium acetate was added to 0.3 M, the sample was split and ethanol added at 3X volume to each aliquot and stored at -20ºC.
Label
biotin
Label protocol
Labeling was performed as described in Kuhn et al., A novel, high-performance random array platform for quantitative gene expression profiling. Genome Res 2004, 14: 2347-2356.
Hybridization protocol
Array hybridization, washing and staining was performed as described in Kuhn et al., A novel, high-performance random array platform for quantitative gene expression profiling. Genome Res 2004, 14: 2347-2356.
Scan protocol
Scanning and image acquisition was performed as described in Kuhn et al., A novel, high-performance random array platform for quantitative gene expression profiling. Genome Res 2004, 14: 2347-2356.
Description
Data extraction and processing was performed as described in Kuhn et al., A novel, high-performance random array platform for quantitative gene expression profiling. Genome Res 2004, 14: 2347-2356.
Data processing
VALUE was generated using a global background subtraction and rank-invariant normalization algorithm, averaging three replicate arrays. Note that in the analysis that will be submitted after receiving the submission number from GEO, we dropped on of the GM11523 arrays beacuse of a within cell individual cell line linear r2 correlation for all genes of <95%, while all remaining within individual cell line correlations were ~99%. To recreate the data file that we used, one would need to drop the ungrouped (unique) normal probe file data from GM11523_56_2_C_H07, and use the remaining two GM11523 arrays, GM11523_36_2_S_D05 and GM11523_46_2_L_F06 and average the probe values from these two files.
