lysed tissue was centrifuged at 500 × g for 5 min at 4 °C, then washed twice more with wash buffer and filtered through 70 µm and then 40 µm cell strainer separately. The pellet was resuspended in 2 ml wash buffer and mixed with 3.6 ml Sucrose Cushion Buffer I (nuclei PURE prep isolation kit, Sigma) containing 1 U/µl RNase inhibitor. Two milliliters of Sucrose Cushion Buffer I with 1 U/µl RNase inhibitor was added into one 15 ml Beckman Coulter centrifuge tube. After that, the 5.6 ml nuclei suspensions were gently added to the top of Sucrose Cushion Buffer I without mixing, followed by centrifuging at 13,000 × g (Beckman Coulter ultracentrifuge) with rotor SW40Ti for 45 min at 4 °C. The purified nuclei pellet was washed by centrifuging at 300 × g for 5 min at 4 °C with wash buffer, and the washed nuclei pellets were resuspended in wash buffer to target ~1000 nuclei/μl.
