Illumina HiSeq 2000 sequencing; Salt augments macrophage-driven host defense in the skin
Salt augments macrophage-driven host defense in the skin
Primary bone-marrow-derived murine macrophages were grown in RPMI1640 (GIBCO) supplemented with 10% FCS, 10mM HEPES, 1% Penicillin-Streptomycin, 5µM beta-mercaptoethanol at a density of 40 million cells per 150cm(2) cell culture flask. After attachment for 2h cells were stimulated with 10ng/ml LPS (Sigma-Aldrich) [M(LPS)] for 24h. 40mM NaCl were added where applicable. 40 million macrophages per group were used to isolate nuclei by centrifugation through a dense sucrose cushion. Subsequently, samples were digested with micrococcal nuclease (Sigma-Aldrich) to generate native chromatin templates consisting mainly of mononucleosomes. Abundance of mononucleosomal DNA was confirmed by gel electrophoresis. Chromatin was extracted with RIPA buffer and cellular debris removed by centrifugation. Subsequently, chromatin was precipitated over night at 4°C with anti-H3K4me3 (NEB  #9751 S) bound to Dynabeads® Protein A (Invitrogen #10002D). MinElute PCR Purification Kit (QIAGEN) was used to purify immunoprecipitated DNA after Proteinase K (Sigma-Aldrich) digestion at 65°C for 4h. 50ng of purified DNA was used for library preparation with the TruSeq ChIP sample preparation kit (Illumina) according to the manufacturer's instructions.
Experimental Factor: stimulus
10ng/ml LPS 24h
Experimental Factor: compound
none
LIBRARY_STRAND
not applicable