GSM8728190: spinal cord,Gpr37l1+/+,Naïve; Mus musculus; RNA-Seq
Fresh SDH tissues from Gpr37l1+/+ and Gpr37l1-/- mice (naive and 1 day after SNI) were microdissected for nuclei isolation according to the previous report. Microdissected SDHs were homogenized in 0.2 ml RNAase-free lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc2, 0.1 mM EDTA, 10 mM Tris-HCl, 1 mM DTT and 0.1% Triton X-100 in diethylpyrocarbonate (DEPC)-treated water) using homogenizer from 10X Genomics nuclei isolation kit (PN-1000493) on ice. The homogenate was loaded into a 3.5-ml polycarbonate ultracentrifuge tube (349622, Beckman-Coulter), and 1.8 ml of sucrose solution (1.8 M sucrose, 3 mM MgAc2, 1 mM DTT, and 10 mM Tris-HCl in DEPC-treated water) was added to the bottom of the tube with the homogenate, and the tube was centrifuged at 24400 RPM for 2.5 h at 4 °C. After removing the supernatants, pellets containing the nuclei were incubated in 100 μl of DEPC-treated water-based PBS for 20 min on ice before resuspending the pellets using the same solution. Nuclei were counted using a hemocytometer and single-nucleus capture was performed using the 10X Genomics Single-Cell 3′ system (v3.1). Single-nucleus libraries from individual samples were pulled and sequenced on the MGISEQ-2000 machine. RNA integrity analysis was performed using the Agilent 2100 Bioanalyzer.