title: Exp2-Irf5-WT-120m-R1
organism: Mus musculus
description: Protocols: For the generation of M1 macrophages differentiated with GM-CSF, bone marrow of wild type and IRF5 knock out C57Bl6 mice was cultured in RPMI-1640 medium (PAA Laboratories) supplemented with recombinant mouse GM-CSF (20 ng/ml; Preprotech). Input samples were processed exactly as the other samples but without the immunoprecipication step. After 8 d, cells were washed with PBS and replated, then stimulated with lipopolysaccharide (LPS - 100 ng/ml; Alexis Biochemicals) for 0 or 2 h. 300 million GM-CSF derived M1 macrophages were used for the IRF5 ChIP experiment. Cells were fixed for 10 minutes with 1 % formaldehyde, quenched with 125 mM of Tris pH 7.5 and washed with ice-cold PBS. Nuclear lysates were isolated as described previously (De Santa et al. 2007) and sonicated with a Bioruptor (Diagenode) to obtain chromatin fragment sizes that average 500bp. Each lysate, but for the input one, was immunoprecipitated with 10 microgram of the following antibodies: IRF5 (Abcam; ab21689). ChIP was performed for each antibody as described previously (Ghisletti et al. 2010). ChIPped DNA was quantified with the Quant-iT dsDNA High Sensitivity Assay Kit (Invitrogen #Q33120). DNA yields ranged from 10-20 ng. 300 million GM-CSF derived M1 macrophages were used for the IRF5 ChIP experiment. Cells were fixed for 10 minutes with 1 % formaldehyde, quenched with 125 mM of Tris pH 7.5 and washed with ice-cold PBS. Nuclear lysates were isolated as described previously (De Santa et al. 2007) and sonicated with a Bioruptor (Diagenode) to obtain chromatin fragment sizes that average 500bp. Each lysate, but for the input one, was immunoprecipitated with 10 microgram of the following antibodies: IRF5 (Abcam; ab21689). ChIP was performed for each antibody as described previously (Ghisletti et al. 2010). ChIPped DNA was quantified with the Quant-iT dsDNA High Sensitivity Assay Kit (Invitrogen #Q33120). DNA yields ranged from 10-20 ng.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-06-20T17:01:59Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T18:21:10Z
attributes.tag: External Id
attributes.value: SAMEA2604496
attributes.tag: INSDC center name
attributes.value: University of Oxford
attributes.tag: INSDC first public
attributes.value: 2014-06-20T17:01:59Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T18:21:10Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2661:Exp2-Irf5-WT-120m-R1
attributes.tag: age
attributes.value: 2 hour
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: M1 Macrophages
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genetic modification
attributes.value: wild type
attributes.tag: genotype
attributes.value: wild type
attributes.tag: sample name
attributes.value: E-MTAB-2661:Exp2-Irf5-WT-120m-R1
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: specimen with known storage state
attributes.value: fresh specimen
attributes.tag: strain
attributes.value: C57BL6