Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

Affymetrix GeneChip Human Genome HG-U133B [HG-U133B]

IL-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells

IL-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells.

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We purified five subsets representing the main stages of human precursor-B-cell differentiation and CD34+lin- cord blood cells. The immunoglobulin (Ig) gene rearrangement status was determined using TaqMan quantitative PCR and GeneScan analysis. To gain more insight in the networks of genes that initiate and/or regulate the different types of Ig gene rearrangements, we  analyzed their gene expression profiles by correlating the initiation of Ig gene rearrangements with specific upregulation of transcription factors. In addition to previously described transcription factors, we identified 16 candidate genes involved in initiation and/or regulation of Ig gene rearrangements.

CellType

Transcription profiling of human precursor-B-cell differentiation

We purified five subsets representing the main stages of human precursor-B-cell differentiation and CD34+lin- cord blood cells. The immunoglobulin (Ig) gene rearrangement status was determined using TaqMan quantitative PCR and GeneScan analysis. To gain more insight in the networks of genes that initiate and/or regulate the different types of Ig gene rearrangements, we  analyzed their gene expression profiles by correlating the initiation of Ig gene rearrangements with specific upregulation of transcription factors. In addition to previously described transcription factors, we identified 16 candidate genes involved in initiation and/or regulation of Ig gene rearrangements.


Affymetrix:Protocol:Hybridization-EukGE-WS2v3

Title: Affymetrix EukGE-WS2v3 Hybridization. Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4_450

Title: Affymetrix EukGE-WS2v4_450 Hybridization. Description:

Affymetrix:Protocol:Hybridization-FlexGE-WS2v3

Affymetrix:Protocol:Hybridization-ProkGE-WS2v3

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-9564

RNA was isolated using RNeasy columns (Qiagen, Hilden, Germany) according to the manufacturers protocol. A first cycle of amplification was performed according to the GeneChip Eukaryotic small sample target labeling assay version II (affymetrix). After this a second round of amplification was performed as described in LABELPTCL....

P-MEXP-9565

An RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY) was used for the production of biotin-labeled cRNA targets by in vitro transcription from T7 RNA polymerase promoters, purified with Qiagen RNeasy Mini Kit (Qiagen, Chatsworth, California) and fragmented by incubation at 94 C for 35 min. 

P-MEXP-9566

11 ug  of cRNA was fragmented for 35 min at 95 degrees C. 10 ug of fragmented cRNA was then hybridized to human genome U133A or U133B microarrays (Affymetrix) for 16 hours at 45 degrees C Hybridization of the arrays was done in an Affymetrix-Genechip Hyb Oven 640. 

P-MEXP-9594

The probe intensity level background was removed using robust multichip analysis (RMA, (reference 1 below)). Probe intensity levels were then quantile normalised (reference 2 below). Array groups corresponding to the development stages were compared based on the perfect match (PM) probe intensity levels only (reference 1 below), by performing a per-probe set two-way analysis of variance(ANOVA, with factors "probe" and "stage"). This results in average expression levels over the biological repeats for each probe set in each stage, as well as a p-value for the significance of the difference between the stages.  reference 1: Irizarry, R.A., B. Hobbs, F. Collin, Y.D. Beazer-Barclay, K.J. Antonellis, U. Scherf, and T.P. Speed. 2003. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:249-264.  reference 2: Bolstad, B.M., R.A. Irizarry, M. Astrand, and T.P. Speed. 2003. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19:185-193.

Transcription profiling of human precursor-B-cell differentiation

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