Illumina HiSeq 2500 paired end sequencing; proj008_chipseq_experiment2
proj008_chipseq_experiment2
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.
Experimental Factor: genotype
wild type
Experimental Factor: immunoprecipitate
IRF5
Experimental Factor: time
2 hour