GSM8392291: NeuN- cells from LPA-treated brain, rep 3; Mus musculus; RNA-Seq
Mice injected with LPA or vehicle were perfused with Hanks' Balanced Salt Solution (HBSS) and RNAse inhibitors (0.2%) (Takara). Whole brains (except for the olfactory bulbs and the cerebellum) were stored at -80° C and were removed from frozen storage and immediately submerged in 1 ml of nuclei isolation buffer (20 mM Tris, 320 mM sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 0.1 mM EDTA, 0.1% Triton-X 100, 0.2% RNase inhibitors, (Takara Bio recombinant RNAse Inhibitor). Extracted nuclei were washed twice in PBS + 0.25mM EGTA + 1% BSA + 0.2% RNase inhibitors (Takara Bio, Mountain View, CA). They were then suspended in PBS + EGTA (250µM) + 1% BSA + 0.2% RNase inhibitors + 1.25 µg/ml 4',6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO) + NeuN antibody (1:500, Abcam), to label neuronal populations. Stained nuclei were sorted on a FACSAria Fusion (BD Biosciences, Franklin Lakes, NJ) gating for NeuN- populations, and collecting non-neuronal nuclei. Sorted nuclei were diluted to ~700-1,500 nuclei/ml, and a final concentration was determined using a fluorescent cell counter. The 10x Genomics Single Cell 3' v3 kit was then used to prepare samples targeting 10,000 profiled cells.