GSM9490620: cKO-PR; Mus musculus; RNA-Seq
Isolation of nuclei from frozen tissue was performed as described by the Allen Institute for Brain Science. All steps performed in 4°C. Briefly, tissues were homogenized in 2ml chilled homogenization buffer (10 mM Tris pH 8, 250 mM Sucrose, 25 mM KCl, 5 mM MgCl2, 0.1 mM DTT, 1x Protease inhibitor cocktail, 0.2 U/μl Protector RNase Inhibitor, 0.1% Triton X-100) and filtered through 70 µm and 30 µm cell strainers. Nuclei pellets were collected by 10 min, 900 x g suspension, and then resuspended in 250 μl chilled homogenization buffer. Finally, debris was removed by Iodixanol 25-29%, and nuclei pellet was resuspended. For neuron specific- single nuclei RNA-sequencing, nuclei pellet was resuspended in 500 μl blocking buffer (1x PBS, 1% BSA, 0.2 U/μl RNase inhibitor) for fluorescent-activated nuclei sorting. Library was performed according to manufacture's description (single-cell 3' protocol, 10x genomics).