GSM6190708
A cerebellum was placed in 5 ml ice-cold nuclei extraction buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 0.1 mM EDTA, 10 mM Tris-HCl and 0.1% Triton X-100) with freshly added 50 μl protease inhibitor cocktail (P8340, Sigma-Aldrich), 5 μl of 100 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich) and 5 μl of 1 M 1,4-dithiothreitol (DTT, Sigma-Aldrich). In addition, 7.5 μl of 40 U/μl RNase inhibitor (N2611, Promega) was also added when mRNA-seq was conducted. Once thawed, the tissue was homogenized by slowly doucing for 15 times with a loose pestle (D9063, Sigma-Aldrich) and 25 times with a tight pestle (D9063, Sigma-Aldrich). The homogenate was filtered with 40 μm nylon mesh cell strainer (22363547, Fisher Scientific) and then transferred into a 15 ml centrifugation tube. The sample was centrifuged at 1000 g for 10 min. The supernatant was removed and the cell pellet was gently resuspended in 1 ml cold nuclei extraction buffer with freshly added 10 μl protease inhibitor cocktail, 1 μl of 100 mM PMSF and 1 μl of 1 M DTT. 1.5 μl of 40 U/μl RNase inhibitor was mixed with the nuclei extraction buffer when mRNA-seq was conducted. The 1 ml sample suspension was split into two 1.5-ml micro-centrifuge tubes (500 μl in each tube). Each sample (500 μl) was gently mixed with 0.75 ml of 50% iodixanol to yield 30% iodixanol solution. 50% iodixanol was prepared by adding 0.4 ml diluent (150 mM KCl, 30 mM MgCl2, and 120 mM Tris-HCl, pH 7.8) to 2 ml of 60% iodixanol (D1556, Sigma). The samples were then centrifuged at 10,000 g for 20 min before the supernatant was removed. The nuclei in each tube were incubated on ice for 10 min in 0.5 ml Dulbecco's phosphate-buffered saline (14190144, Life Technologies) containing 2.0% normal goat serum (50062Z, Life Technologies). The nuclei were resuspended and then pooled together (generating ~1 ml in total). Nuclei were sequenced using the 10x Genomics Chromium Single Cell 3' kit v2 (10x Genomics) according to the manufacturer's suggested protocol for nuclei.