GSM9497730: WT-Veh; Mus musculus; RNA-Seq
Tissue samples were subjected to lysis in 1 ml of lysis buffer (10 mM Tris-HCl, 5% Nonidet P40, 3 mM MgCl2, and 10 mM NaCl) along with 1 U/μl RNasin Plus RNase Inhibitor (Promega) for 15 minutes in a douncing tube placed on ice. The tissue was then homogenized using a loose pestle with ten dounces. The resulting suspension was filtered through a 70 μm mesh and centrifuged at 500 g for 5 minutes at 4°C. Post-lysis, the sample was treated with 2 ml of ice-cold nucleus wash buffer (5% wt/vol BSA, 0.25% wt/vol glycerol, 40 U/mL Protector RNAse inhibitor, 0.5X PBS), passed through a 20 μm mesh twice, and re-centrifuged under the same conditions. The supernatant was discarded, and the nuclear pellet was resuspended in 500 μl of wash buffer. An additional 500 μl of 50% (wt/vol) iodixanol (Optiprep) working solution was added to aid in further cleaning. This suspension was then layered over 1 mL of 29% (wt/vol) iodixanol solution in a 2 mL centrifuge tube and centrifuged at 10,000g for 30 minutes at 4°C. Finally, the nuclei were resuspended in a suitable volume of nuclei buffer to achieve a concentration of 500-1,000 nuclei/μl for subsequent capture. Single nucleus RNA sequencing libraries were prepared using the Chromium Next GEM Single Cell 3′ Gel Bead v4 Kit (10x Genomics)