1.8 m sucrose cushion solution

Approximately 100 mg of tissue was homogenized in cold lysis buffer (Nuclei PURE Lysis Buffer/1 mM dithiothreitol/0.1% Triton X-100; Sigma-Aldrich) in a Dounce homogenizer. The homogenate was transferred to a 50-ml conical tube, vortexed for 2 to 3 s, and incubated for 10 min on ice in a total of 10-ml lysis buffer. Tissue lysate was resuspended with 18 ml of cold 1.8 M Sucrose Cushion Solution and layered slowly over 10 ml of cold 1.8 M Sucrose Cushion Solution (Sigma-Aldrich) in an ultracentrifuge tube (Beckman Coulter) on ice. Samples were centrifuged for 45 min at 30,000g at 4°C. Pelleted nuclei were resuspended in 1-ml cold nuclei suspension buffer (NSB; 0.01% phosphate-buffered saline and 0.1% bovine serum albumin; New England BioLabs) and SUPERase-In RNase inhibitor (Thermo Fisher Scientific) (19 (link)). The nuclei suspension was mixed with an additional 4 ml of cold NSB and centrifuged for 5 min at 500g at 4°C. After a second wash in 5-ml cold NSB, nuclei were resuspended in 100 to 200 μl of cold NSB and counted on an automated cell counter (Bio-Rad). The concentration of the nuclei suspension was adjusted to ~1000 nuclei/μl.