GSM8169937: Nuclei rep2 [n2_S2]; Mus musculus; miRNA-Seq
For nuclei extraction,Triton X-100  (1% in the above buffer) was added to homogenate (0.5:1 v/v). Sample was stirred on a vortex mixer for 30 s and the 1.5 M sucrose in the above buffer was added (0.25:1 v/v). After centrifugation at 2000g × 10 min, the pellet containing the nuclei was washed twice with 0.1M Barnes solution containing 0.085 M KCl, 0.0085 NaCl, 0.0025 M MgCl2, trichloroacetic acid-HCl 0.005 M. For nuclei lipid microdomain extraction, HN9.10 cell nuclei were washed in 10 mM Tris, containing 0.25 M sucrose, 2.5 mM MgCl2, 0.5 mM PMSF, and 1% Triton X-100, pH 7.5, at 0 °C, in order to remove the external nuclear membrane preserving the inner nuclear membrane, and were centrifuged at 1000g for 15 min at 4 °C. The pellet was re-suspended in 0.1 M Barnes solution  at pH 7.4, and was digested with DNase I (120 μg/ml) and RNase cocktail (500 μg/ml) for 15 min at 37 °C. The reaction was stopped on ice with Barnes solution (1:1 by vol.). After centrifugation at 5000 rpm for 5 min, the pellet was resuspended in 30 mM Tris containing 0.4 M ammonium sulphate, which facilitated the RNA precipitation, and 1 mM PMSF, pH 8.4, and then was centrifuged at 10,000 rpm for 15 min. Paired-end