GSM8606603: 16mo_replicate_2; Mus musculus; RNA-Seq
From each mouse, both snap frozen femoral sciatic nerves were thawed in nuclei isolation media (NIM, 250 mM Sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl) with 25 mM DTT, 1X protease inhibitor (05056489001, Roche), 0.1% Triton X-100 and RNase inhibitor (N2515, Promega). Each pair of nerves was homogenized using a Kimble dounce tissue grinder (D8938, Merck) and the resulting suspension was filtered through a 30 μm CellTRics (Sysmex, MSA150914) into a LoBind Eppendorf tube (Eppendorf). Nuclei were pelleted (1000 x g, 10 min, 4oC) and washed with NIM + DTT + Protease inhibitor (05056489001, Roche) and RNase inhibitor (N2515, Promega). After the wash, nuclei were resuspended in sorting buffer (1 mM EDTA, 0.2 U/μL RNase inhibitor (N2515, Promega), 2% fatty acid-free BSA (A7030-100g, Sigma) in PBS). Finally, the nuclei were filtered again through a 30 μm CellTRics (Sysmex, MSA150914), and DRAQ7 (#7406, Cell Signalling) was added to the nuclei suspension for nuclei detection by flow cytomtery. 10,000-20,000 nuclei were sorted using a S3 Fluorescence-activated cell sorter (Bio-Rad). Nuclei were resuspended and used for snRNA-seq library generation following the Single Cell 3' Reagents Kits V3.1 User Guidelines (10x Genomics).