H3K9me2_ChIPSeq_control4
Rattus norvegicus
source_name
control 4
cell type
PCM1-positive nuclei from left ventricle of the heart
strain
Sprague-Dawley
chip antibody
anti-H3K9me2, clone MABI 0307 (Active Motif, La Hulpe, Belgium)
hypertrophy induction
Male Sprague Dawley rats of ~280 g not assigned to the exercise protocol were used as control.
isolation of material from sorted nuclei
A solution of at least 100000 freshly sorted nuclei for nucleosome extraction was topped up with MNase buffer (0.32 M sucrose, 4 mM MgCl2, 1 mM CaCl2 and 1 µL/mL cOmplete PI cocktail) to a final volume of 1.5 mL and spun for 10 minutes (1,000×G, 4°C). All but 100 µL supernatant was removed, and nuclei were gently suspended in an additional 1.4 mL MNase buffer and spun for 10 minutes (1,000×G, 4°C). All but 100 µL supernatant was again removed, and the pelleted nuclei were suspended to a concentration of 4,000 nuclei (as counted by flow cytometry) per µL in MNase buffer. Micrococcal nuclease (Affymetrix USB, Staufen, Germany) was subsequently added (20 U/mL) to the nuclei suspension. In order to digest the chromatin to a predominantly mononucleosomal resolution, the reaction was stopped after 8 minutes of incubation at 37°C by transfer on ice and by the addition of ½ volume of nucleosome extraction buffer (15 mM EDTA, 0.3% Triton X-100, 10 mM Na2HPO4, 3.5 mM KH2PO4, 1.5 M NaCl, 20 µg/mL and 3 µL/mL cOmplete PI cocktail at pH 7.4). Nucleosomes were subsequently extracted at 4°C overnight on a vertical rotor (30 RPM). The next day, nuclear debris was removed by centrifugation for 10 minutes (21,000×G, 4°C), after which 10 µL Protein G Dynabeads (Life Technologies) were added to the supernatant for preclearing the solution and removing the remainder of the antibodies that were used for nuclei labelling. After 1 hour at 4°C on a vertical rotor (30 RPM), the beads were removed by magnetic separation, and the solution split in 2 equal fractions. Per volume, 2.5 volumes of ChIP buffer (3 mM Na2HPO4, 1 mM KH2PO4, 50 mM NaCl, 5 mM EDTA, 2 µg/mL BSA and 1 µL/mL cOmplete PI cocktail at pH 7.4) were added, as well as per fraction either the mouse monoclonal anti-H3K9me2 (clone MABI 0307; Active Motif, La Hulpe, Belgium) or the rabbit polyclonal anti-H3K27me3 (07-449; Millipore, Watford, UK) antibody, each at 1 µg/mL. These solutions were kept overnight on a vertical rotor at 4 °C (5 RPM) to allow for antibody binding. On the next day, 20 µL Protein G Dynabeads were added to each solution, and after mixing on a vertical rotor at 4°C (5 RPM) for 2 hours, beads were magnetically separated and the cleared solution transferred to a new tube to be kept as input fraction. Beads were washed 8 times with RIPA buffer (50 mM Tris-HCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate, 0.5 M LiCl and 1 µL/mL cOmplete PI cocktail at pH 7.5) and once with TE buffer (50 mM Tris-HCl, 1 mM EDTA and 1 µL/mL cOmplete PI cocktail at pH 7.5), after which beads were resuspended in 200 µL TE and transferred to a new tube. Proteinase K (2 mg) was subsequently added to each tube (including input fractions), and after incubation at 42°C for 30 minutes, DNA was extracted using the QIAquick PCR purification kit (QIAGEN, Manchester, UK) as follows: binding buffer PB with pH indicator was added directly to the suspension, pH adjusted using Na(CH3COO) and after an incubation of 10 minutes (and magnetic separation of the beads from the ChIP samples), the DNA binding, washing and elution in 50 µL water was done as per the kit’s instructions. Purified DNA was rendered blunt ended using the NEBnext End Repair Module (New England Biolabs) as per the manufacturer’s instructions.