Then, the supernatant was removed and 300 µl of 2%(w/v) normal goat serum (50062Z, Life technologies) in Dulbecco’s PBS (DPBS, Life technologies) was added to resuspend the nuclei pellet. The mixture was centrifuged at 10,000 RCF for 20 min. 500 µl of the sample was mixed with 750 µl of 50%(w/v) iodixanol (made by mixing 4 ml of OptiPrepTM gradient (Sigma-Aldrich) and 0.8 ml of diluent ). The supernatant was discarded, and the pellet was resuspended in 0.5 ml of ice-cold NEB with freshly added 5 µl of PIC, 0.5 µl of PMSF, 0.5 µl of DTT, and 0.75 µl of RNase inhibitor. The cell suspension was centrifuged at 1000 RCF for 10 min. The homogenate was filtered with a 40 µm cell strainer (22-363-547, Thermo Fisher Scientific) and collected in a 15-ml centrifuge tube. The tissue was homogenized in tissue grinder (D9063, Sigma-Aldrich). One piece of mouse frontal cortex tissue (6-10 mg) was placed in 3 ml of ice-cold nuclei extraction buffer (NEB) with freshly added 30 µl of protease inhibitor cocktail (PIC, Sigma-Aldrich), 3 µl of 100 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich) in Isopropyl alcohol, 3 µl of 1 M dithiothreitol (DTT, Sigma-Aldrich), and 4.5 µl of ribonuclease (RNase) inhibitor (2313A,Takara Bio). Nuclei extraction: All steps were conducted on ice, and all centrifugation was conducted at 4 ☌. Neuron from the frontal lobes of the mouse cortexĪntibody: anti-H3K27ac (39135, Active Motif) GEO help: Mouse over screen elements for information.
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