inhibitor was added and mixed well. The lysed tissue was centrifuged at 500 rcf for 5 minutes 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. 2 ml 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, and followed by centrifuging at 13,000 x rpm (30,000 rcf) (Beckman Coulter ultracentrifuge) with rotor SW40Ti for 45 minutes at 4°C. The purified nuclei pellet was washed by centrifuging at 300 rcf for 5 min at 4°C with wash buffer, and the washed nuclei pellets was resuspended in wash buffer to target ~ 1000 nuclei/μl.
