Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of 3 genotypes in mouse

ABSTRACT: For transcription profiling six mouse T cells samples were analyzed: 3 genotypes 2 samples for each genotype were complared. Two wt animals-control;2 animals containing deficiency allele (Df11(1)) on chromosome 11 and 2 with the reciprocal duplication on chromosome 11(Dp11(1)). The part of the chromosome 11 deleted or duplicated is 0.8Mb between Gast and Hcd17b genes. Splenic CD4+ T cells were purified using anti-CD4 MiniMacs beads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's protocol and were plated at 106 cells/ml in complete RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 0.3 mg/ml L-glutamine, 10 micromolar 2-mercaptoethanol, 100 U/ml penicillin, and 0.1 mg/ml streptomycin in 6 well plates coated overnight with anti-CD3 (5 _g/ml) and anti-CD28 (1 _g/ml) antibodies. After 5 days of culture cells were collected and prepared. Cells were suspended at 107 cells/ml in complete medium and cytokine secretion was induced with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 µg/ml ionomycin (Sigma). After 2 hours protein secretion was inhibited with brefeldin A (1:2000, eBioscience) and after 5 hours RNA from 2x106 cultured CD4+ T cells (RNAeasy kit, Qiagen, Hilden, Germany) was isolated following cytokine induction and 10 mg of RNA was used for hybridiation to mouse 430 2.0 array, Affymetrix, Santa Clara, CA arrays.

ORGANISM(S): Mus.Musculus

SUBMITTER: Olga Ermakova

PROVIDER: E-MTAB-447 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In this work we present an analytical strategy to systematically identify early regulators by combining gene regulatory networks (GRN) with GWAS. We hypothesized that early regulators in T-cell associated diseases could be found by defining upstream transcription factors (TFs) in T-cell differentiation. Time series expression and DNA methylation profiling of T-cell differentiation identified several upstream TFs, of which TFs involved in Th1/2 differentiation were most enriched for disease associated SNPs identified by GWAS. NaÃ¯ve CD4+ T cells were isolated from buffy coat of four healthy donors using a naÃ¯ve CD4+ T cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). NaÃ¯ve CD4+ T cells were stimulated with plate-bound anti-CD3 (500 ng/mL), soluble anti-CD28 (500 ng/mL), in the presence of IL-12 (5 ng/mL), IL-2 (10 ng/mL) and antiâ??IL-4 (5 Âµg/mL) for Th1, IL-4 (10 ng/mL), IL-2 (10 ng/mL) and anti-IL-12 (5 Âµg/mL) and antiâ??IFN-g (5 Âµg/mL) for Th2. Cells were cultured for six days in Iscoveâ??s modified Dulbecco medium (IMDM) supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 10% heat-inactivated FCS (PAA Laboratories, Linz, Austria), 5 ÂµM Î²â??mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 ug/mL gentamicin (Sigma-Aldrich, St. Louis, Missouri, USA). Cells were cultured for 6 days and then re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of corresponding polarizing cytokines and antibodies for another 2 days (Zhang et al. 2013). RNA was extracted using a miRneasy Mini Kit (Qiagen). The RNA concentrations were analysed with NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). For the gene expression microarray analysis, The cRNA was prepared using a Low Input QuickAmp Labeling Kit. For Th1 and Th2 cells the gene expression microarray analysis was performed using SurePrint G3 Human Gene Expression 8x60K microarray kit, according to the manufactureâ??s instruction (Agilent Technologies).

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Transcription profiling of 3 genotypes in mouse

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