Nuclei Isolation from Tissue Samples

Nucleus isolation was done as previously described^{8 (link)}. Briefly, tissue samples were cut into pieces <0.5 cm and homogenized using a glass Dounce tissue grinder (Sigma, cat. no. D8938). The tissue was homogenized 25 times with pestle A and 25 times with pestle B in 2 ml of ice-cold nuclei EZ lysis buffer. The sample was then incubated on ice for 5 min, with an additional 3 ml of cold EZ lysis buffer. Nuclei were centrifuged at 500g for 5 min at 4 °C, washed with 5 ml ice-cold EZ lysis buffer and incubated on ice for 5 min. After centrifugation, the nucleus pellet was washed with 5 ml nuclei suspension buffer (NSB; consisting of 1× PBS, 0.01% BSA and 0.1% RNase inhibitor (Clontech, cat. no. 2313A)). Isolated nuclei were resuspended in 2 ml NSB, filtered through a 35 μm cell strainer (Corning-Falcon, cat. no. 352235) and counted. A final concentration of 1,000 nuclei per µl was used for loading on a 10x channel.