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E-MTAB-7076

ABSTRACT: Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach

PROVIDER: E-MTAB-7076 | ExpressionAtlas | 2018-09-17

REPOSITORIES: ExpressionAtlas

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			Action	DRS
	E-MTAB-7076-analysis-methods.tsv	Tabular
	E-MTAB-7076-analytics.tsv	Tabular
	E-MTAB-7076-atlasExperimentSummary.Rdata	Rdata
	E-MTAB-7076-configuration.xml	Xml
	E-MTAB-7076-g2_g1-mvaPlot.png	Other

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Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach

Project description:Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach

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Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach

Project description:Alpk1-deficient mice demonstrate exacerbated colitis and increased IL-12/Th1 response upon challenge with an intestinal pathobiont, Helicobacter hepaticus (Hh). Hematopoietic compartment is driving the pathogenic phenotype in this animal model, and Alpk1 is highly expressed in myeloid cells (macrophages and dendritic cells). Alpk1 deficiency has a recessive phenotype, since Alpk1+/- (heterozygous) mice show the same phenotype as the wild type mice. Mouse bone-marrow derived macrophages (BMDMs) show elevated IL-12 production in Alpk1-/- mice in response to stimulation with Hh. Since the molecular mechanism of how Alpk1 deficiency affects macrophage response to Hh is unknown, we aimed to characterise global changes in gene expression in Alpk1+/- vs Alpk1-/- bone-marrow differentiated cells (BMDMs). Cells were isolated from bone marrow of Alpk1+/- and Alpk1-/- (mixture of bone marrows from three mice per genotype) and plated in BMDM differentiation medium (RPMI, 10% FCS, penicillin and streptomycin, 50 micro beta-mercapthoethanol, 20 ng/ml recombinant mouse GM-CSF(Peprotech)), 7 million cells in per 10 sm uncoated TC dish in 10 ml of medium for eight days, extra 10 ml of medium was added to plates at day 4, before collection and replating in 96-well plates, 150 thousand cells/200 microliters of differentiation medium per well in technical triplicates per genotype/stimulation condition (R1-R3 labels of the samples). The following day BMDMs were stimulated with MOI of 10 of Hh and 10ng/ml of mouse IFNg (Peprotech) (Alpk1+/- BMDMs – het _Hh_8h vs Alpk1-/- BMDMs – alpk1_Hh_8h) or IFNg only (het _nonstim_8h vs alpk1_nonstim_8h) before lysis for RNA extraction using Quick-RNA kit from Zymo Research. Purified RNA was submitted to the Welcome Trust Centre for Human Genetics (Oxford) for RNA-Sequencing

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