GSM7678198: iPSC-derived dopamine neurons, normal control, co-cultured with astrocytes, 2D (plate) format; Homo sapiens; RNA-Seq
All steps were performed on ice.  Wide-bore Rainin LTS pipette tips were used exclusively throughout. Frozen cell pellets were thawed on ice, resuspended in cold homogenization buffer (5mM MgCl2, 3mM Mg(Ac)2, 10mM Tris pH7.8, 0.017 mM PMSF, 0.17 mM b-mercaptoethanol, 320 mM Sucrose, 0.1 mM EDTA, 0.1% NP40, RNAse inhibitors, and Protease inhibitors), and transferred to a 2 ml glass dounce.  The tissue was manually lysed with 50 strokes each of the "A" and "B" pestles.  Homogenate was filtered using 70 um Flowmi pipette tip filters and gently mixed 1:1 with a 50% Iodixanol solution (5mM MgCl2, 3mM Mg(Ac)2, 10mM Tris pH7.8, 0.017 mM PMSF, 0.17 mM b-mercaptoethanol, RNAse inhibitors, Protease inhibitors, and 50% Iodixanol).  A centrifugation gradient was set up using 600 ul 40% Iodixanol solution (5mM MgCl2, 3mM Mg(Ac)2, 10mM Tris pH7.8, 0.017 mM PMSF, 0.17 mM b-mercaptoethanol, RNAse inhibitors, Protease inhibitors, 160 mM Sucrose, 0.2 ug/ul OrangeG, and 40% Iodixanol), 600 ul 29% Iodixanol solution (5mM MgCl2, 3mM Mg(Ac)2, 10mM Tris pH7.8, 0.017 mM PMSF, 0.17 mM b-mercaptoethanol, RNAse inhibitors, Protease inhibitors, 160 mM Sucrose, and 29% Iodixanol) and 800 ul of the 50% Iodixanol + Sample mixture (25% Iodixanol final concentration).  This was centrifuged at 3000 x g for 1 hour with the breaks disengaged.  Any debris on the top surface was removed, and 200 ul of the thin cloudy layer containing the nuclei was extracted, mixed with 1.8 ml PBS + 1% BSA solution, and centrifuged at 500 x g for 10 minutes with breaks engaged.  1.8 ml supernatant was removed and the remaining 200 ul plus nuclei pellet was resuspended in 1.8 ml PBS + 1% BSA solution.  The nuclei suspension was counted via hemocytometer and checked for nucleus quality.  A portion of this suspension was then diluted to achieve the correct nuclei concentration for the 10x Chromium NextGEM 3' protocol with a target of ~4,000 nuclei per sample. Standard 10x protocol found in "Chromium NextGEM Single Cell 3' Reagent Kits v3.1 User Guide, Rev D" (single index)