Illumina HiSeq 2000 sequencing; Identification of CaMKII-dependent H3S28p target genes
Identification of CaMKII-dependent H3S28p target genes
Mouse hearts are perfused in a Langendorff apparatus and digested via liberase solution (Roche), the cells are going through different Ca2+ containing solutions. Cells are subcequently plated in a cellculture dish. The cells get fixated via incubation of 1% formaldehyde for 10 min at RT. The reaction is quenced using Glycin at a final concentration of 250 mM. Finally, the cells get collected and centrifuged to a cell pellet. The cells are lysed with the following buffers: Buffer1: 50mM Hepes‐KOH pH7.5, 140 mM NaCl, 1mM EDTA, 0.2ml 10% glycerol, 0.5% NP‐40, 0.25% Triton, X‐100 Buffer2: 200mM NaCl, 1mM EDTA (pH 8), 0.5mM EGTA (pH 8), 10mM Tris (pH 8) Buffer3: 50mM Tris (pH 8), 0,1% SDS, 0,95 % NP40, 0,1% Na-deoxycholate, 10mM EDTA, 150mM NaCl. The chromatin is fragmented via bioruptor sonification (Diagenode) using 4x10 cycles of sonification. The dna is precipitated using phenol/chloroform/isoamylalcohol and PhaseLock Tubes (5prime). Libraries are prepared according to the manufacture's protocol (NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina)
Experimental Factor: immunoprecipitate
H3S28P (#9713, Cell Signaling)
Experimental Factor: genotype
wild type genotype
Experimental Factor: stimulus
isoprenaline