Illumina HiSeq 2000 sequencing; High salt reduces the activation of IL-4+IL-13 stimulated macrophages
High salt reduces the activation of IL-4+IL-13 stimulated macrophages
Primary bone-marrow-derived murine macrophages were grown in RPMI1640 (GIBCO) supplemented with 10% FCS, 10mM HEPES, 1% Penicillin-Streptomycin, 5uM 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)] or 10ng/ml IL-4 (R&D Systems) and 10ng/ml IL-13 (Invitrogen) 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 4C with anti-H3K4me3 (NEB  #9751 S) or anti-H4Ac (Millipore #06-866) 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 65C 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: compound
NaCl
Experimental Factor: dose
40 millimolar
Experimental Factor: immunoprecipitate
anti-H4ac (Millipore #06-866)
LIBRARY_STRAND
not applicable