individual: Control9 disease state: control tissue: blood cell type: Sorted B cell
Treatment protocol
Peripheral blood was drawn into heparin-containing sterile tubes and peripheral blood mononuclear cells were prepared by Ficoll (Amersham) density gradient centrifugation for immediate use. B cells were labeled with a biotin anti-CD19 monoclonal antibody (HIB19 clone, Pharmingen) at 4°C and revealed by phycoerythrin-labelled streptavidin (Biomeda) before immediate B cell sorting with high speed cell sorter (FACS Diva, Beckton-Dickinson).
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized on Affymetrix GeneChip human genome U133 plus 2.0 (with probe sets representing 38,572 UniGene clusters) according to the manufacturer’s instructions. After hybridization and washings, arrays were stained with PE-conjugated streptavidin (10 μg/ml) before scanning.
Scan protocol
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Description
Gene expression data from human sorted B cells. T09 JLP_T09_U133_2.CEL
Data processing
Raw Affymetrix data were analyzed using R (R Development Core Team, 2008; The Comprehensive R Archive Network: http://cran.r-project.org/) and Bioconductor (Bioconductor: http://www.bioconductor.org/) softwares. The quality of the 27 Affymetrix genechips and RNA was assessed using the Bioconductor AffyPLM and simpleaffy packages, with qc, AffyRNAdeg, fitPLM, image, RLE and NUSE functions. For normalization and background correction. Raw values were pre-processed with RMA or GCRMA (library simpleAffy). The Sample data table shows genes that are considered as expressed by affy (P) at least in one of the patients and controls, thus, reporting ~65% of the data rows in the U133 Plus 2.0 Array.
