ChIPseq_input_control
Rattus norvegicus
source_name
control
cell type
PCM1-positive nuclei from left ventricle of the heart
strain
Sprague-Dawley
chip antibody
NA
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 50 µL was used as input. These solutions were kept overnight on a vertical rotor at 4 °C (5 RPM). 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, 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.