GSM5609934: Specimen_1; Homo sapiens; RNA-Seq
Briefly, 14 mL of sucrose cushion (1.8 M sucrose (Sigma-Aldrich, RNase & DNase free, ultra pure grade), 10 mM Tris-HCl pH 8.0 (Invitrogen), 3 mM MgAc2 (Sigma-Aldrich), protease inhibitor cocktail (Roche)) was added to the bottom of centrifuge tubes (Beckman Coulter). Using a glass homogenizer (Wheaton), a freshly isolated or frozen mouse cortex sample was subjected to dounce homogenization (21 times with loose pestle followed by 7 times with tight pestle) in 12 mL of homogenization buffer (0.32M sucrose, 5 mM CaCl2 (Sigma-Aldrich), 3mM MgAc2, 10 mM Tris-HCl pH 8.0, 0.1% Triton X-100 (Sigma-Aldrich), 0.1 mM EDTA (Invitrogen), protease inhibitor cocktail). For in vitro cultured cells, cell pellets (~ 5 million cells) were resuspended in homogenization buffer and dounced 20 times with a loose pestle. Homogenates (12 mL) were layered onto the sucrose cushion in the centrifuge tubes, and 10 mL of homogenization buffer was added atop of the homogenates. The tubes were then centrifuged in a Beckman Coulter L7-65 Ultracentrifuge at 25,000 rpm at 4C for 2 hr using a Beckman Coulter SW28 swinging bucket rotor (Beckman Coulter). The supernatant was carefully removed via aspiration. 1 mL of chilled DPBS with RNase inhibitor (Lucigen) was added to resuspend the nuclear pellet, and nuclei were subsequently transferred to a 1.5-mL tube. Nuclei were pelleted at 5,000 rpm for 10 min at 4C, and then resuspended in 0.01% BSA (Sigma-Aldrich) in DPBS. After resuspension, nuclei were filtered through a 40-mm cell strainer (Fisher Scientific), visually inspected for morphology and quality assurance, and counted using a Fuchs-Rosenthal counting chamber before droplet microfluidic encapsulation. Drop-seq