GSM7437219: day 3 [snRNA-Seq]; Mus musculus; RNA-Seq
Injured forebrain hemispheres were frozen on dry ice and stored at -80 °C until processing. For each condition, three hemispheres were collected and pooled during the single-nucleus suspension preparation. All steps for nuclei isolation were performed on ice, with instruments and plastics pre-chilled on ice, or centrifugation steps at 4°C. The frozen brain tissues were combined with 2 ml of lysis buffer (10 mM Tris, pH = 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1 % NP-40; 0.2 U/ul RNaseOUT; 0.32 M Sucrose; 1x protease inhibitor, Roche) and transferred to 2 ml Dounce tissue grinder (Sigma). Tissues were homogenized by 20 strokes with pestle A, followed by 20 strokes with pestle B and filtered using 30 um filter (Celltricks). Tissue grinder was rinsed with 1 ml HEB buffer (Hibernate A, ThermoFisher; 10 ul/ml GlutaMAX, ThermoFisher; 0.32 M Sucrose) and the wash filtered into the same tube. Homogenates were split by careful pipetting of 1.2 ml into two 2 ml tubes prefilled with 500 ul sucrose cushion (1.2 M sucrose; 10 mM Tris; 10 mM NaCl; 3 mM MgCl2; 0.2 U/ul RNase OUT) and centrifuged at 13000 g for 20 minutes at 4°C. Supernatants were removed, except 100 ul to keep the pellets covered. Pellets with the remaining supernatant were resuspended in 1 ml sucrose solution (1.0 M sucrose; 10 mM Tris; 10 mM NaCl; 3 mM MgCl2; 0.2 U/ul RNase OUT), pipetted into 2 ml tubes prefilled with 500 ul sucrose cushion and again centrifuged at 13000 g for 20 minutes at 4°C. Supernatants were removed and the pellets resuspended in 1 ml of Nuclei Wash Buffer (DPBS without calcium and magnesium; 0.5 % BSA; 0.2 U/ul RNase OUT; all ThermoFisher) and transferred into 1.5 ml tubes. Samples were centrifuged at 500 g for 5 minutes, the pellets resuspended in 1 ml of Nuclei Wash Buffer and centrifuged again. The final pellets with nuclei were resuspended in 1 ml of Nuclei Wash Buffer and filtered using 30 um filter. Nuclei stained with Propidium Iodide were counted using CellDrop Automated Cell Counter (Denovix) at default settings. Nuclei suspensions were diluted to final concentrations of 1000 nuclei/ul and single-nucleus RNA-Seq (snRNA-Seq) libraries were prepared. Single-nucleus RNA-Seq (snRNA-Seq) libraries were prepared using Chromium Next GEM Single Cell 3' Kit v3.1 (1000268, 10X Genomics) according to the manufacturer's protocol.