All NF1-affected and -unaffected banked lymphoblastoid cell lines (LCLs; Epstein-Barr virus (EBV)-transformed peripheral blood B-cell lymphocytes) from human kindred with neurofibromatosis type 1 were obtained from the Coriell Institute (pedigree 2176; Camden, NJ, USA). Cultures were maintained in an incubator at 37oC with 5% CO2 in T25 flasks in medium with RPMI 1640 supplemented with 2 mM L-glutamine, 100 Units/mL penicillin, 100 mg/mL streptomycin and 15% heat inactivated fetal bovine serum (all tissue culture reagents were from Gibco/Invitogen, Carlsbad, CA, USA). Cultures were grown to a density from 0.9 x 106 to 1.3 x 106 cells/mL and harvested in Trizol reagent (Invitrogen, Carlsbad, CA, USA).
Extracted molecule
total RNA
Extraction protocol
Trizol cell lysates were mixed with chloroform (1/5 of lysate volume), vortexed for one minute and centrifuged in a table-top centrifuge at 13,000 rpm for 15 min at 4ºC. The aqueous phase was used for RNA isolation. The aqueous phase was mixed with equal volume of 70% ethanol and immediately loaded onto RNeasy mini columns (Qiagen, Valencia, CA, USA), with subsequent steps performed as per the manufacturer’s protocol. The RNA quality was estimated on a 2100 Bioanalyzer, RNA 6000 Nano Chips (Agilent, Santa Clara, CA, USA). Samples with RNA integrity number (RIN) of 8.0 and above were used for further analysis.
Label
Cy3
Label protocol
Biotinylated cRNA were prepared with the Illumina TotalPrep RNA amplification kit (Ambion Inc., Austin, TX) per the manufacturer's instuctions.
Hybridization protocol
Amplified biotinylated cRNA (1.5 ug) were hybridized to HumanWG-6_v2 Sentrix BeadChips. In all steps, care was exercised to avoid batch effects. Samples were hybridized to the microarrays at 55ºC for 16-17 hours. Microarrays were washed to remove non-specifically bound cRNA, stained with 1 mg/mL Streptavidin-Cy3 (GE Healthcare, Piscataway, NJ, USA) and dried.
Scan protocol
Microarrays were scanned in BeadStation 500 scanner. Image acquisition and initial image analysis were done with Illumina BeadScan and BeadStudio applications.
Description
13-A
Data processing
Raw data from the Illumina BeadChips was corrected for background and quantile normalized using BeadExplorer (version 1.5.0), a Bioconductor module developed for quality control, normalization, annotation and exploration of Illumina BeadChip data. Average detection value was calculated for each transcript, and the transcripts with average detection values below 0.99 were excluded from further analysis.
Non-normalized data is available in the 'GSE18444_non-normalized_data.txt' file, which is linked to the Series GSE18444 record as a supplementary file.
