title: GSM6568100: Carm1_WT_Input_Rep2; Mus musculus; ChIP-Seq
library_name: GSM6568100
library_construction_protocol: Briefly, mosaic heart that contained AAV9-Cre infected Carm1KO or control AAV9-GFP infected cardiomyocytes was perfused with 20ml 1% PFA and then 20ml 0.25M Glycine to quench the excessive PFA. Then the heart was harvested. To obtain single nuclei suspension, the heart was cut into small pieces and then homogenized with an electric tissue homogenizer for 30sec on ice. Then the lysate was then washed with 1% BSA/PBS and then filtered through one 70 micrometer strainer and one 40 micrometer strainer. Then the nuclei were stained with PCM1+ antibody (1:500, Sigma, HPA023370) and subjected to FACS to enrich the cardiomyocytes nuclei for H3R17me2a ChIP.  The isolated cardiomyocyte nuclei were incubated in nuclear isolation buffer (Sigma-Aldrich). Then the chromatin was fragmented for 5 min at 37ºC by using MNase before addition of EDTA to stop the reaction. The chromatins were then diluted in complete IP buffer(20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1x Protease inhibitor cocktail, 1 mM PMSF) and incubated at 4 ºC for 1 hour. After the incubation, the chromatins were precleared with Protein A Dynabeads (Invitrogen) and immunoprecipitated with 1 µg H3R17me2a (Active motif, 39707) antibody for overnight. Immunoprecipitated complexes were washed twice with low salt wash buffer I (20 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate, 2 mM EDTA and 150 mM NaCl) and twice with high salt wash buffer (20 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate, 2 mM EDTA and 500 mM NaCl). Protein–DNA complexes were eluted in 30 μl of ChIP elution buffer (100 mM NaHCO3 and 1% SDS) for 2 h at 68 °C. Immunoprecipitated DNA was purified and used for sequencing. The libraries are constructed with Takara ThruPLEX DNA-seq kit.