title: GSM5823254: NeuN+ nuclei from the septal area of adult C57BL6/J mice [snRNA-seq]; Mus musculus; RNA-Seq
library_construction_protocol: Nuclei were prepared as reported before with modifications. Specifically, septal tissues were dissected from 10 mice, pooled together, and stored in 1mL of ice-cold Brain Homogenization Buffer [10 mM Tricine-KOH pH8, 25 mM KCl, 5mM MgCl2, 250 mM Sucrose and 0.1% Triton X-100), supplemented with 0.5 U/mL SUPERase•In (ThermoFisher, AM2696), 0.5 U/mL RNase Inhibitor (ThermoFisher, AM2682) and 2x EDTA-free Halt protease inhibitor cocktail (ThermoFisher, 87786)]. Samples were transferred into a cold Wheaton 1mL glass homogenizer and ground by 10 strokes with the “Loose” pestle, followed by 14 strokes with the “Tight” pestle. Homogenate was then filtered with a 40um cell strainers and underlaid with 1mL 20% iodixanol in a clear ultracentrifuge tube. Sample was centrifuged at 10,000 g for 20 min at 4 oC with low acceleration/deceleration setting. Nuclei was resuspended in 500uL Staining Buffer (1x DPBS, 0.5% BSA, 0.1% Triton X-100, 0.05% NP-40, 0.5 U/mL SUPERase•In, 0.5 U/mL RNase Inhibitor, 2x EDTA-free Halt protease inhibitor cocktail) and stained with anti-NeuN-AF647 (Abcam, ab190565, 1:500) and Hoechst 33342 (ThermoFisher, H3570, 1:500) at 4 oC for 30 minutes.  Nuclei was washed once with Staining Buffer, spun down at 1000g for 5 minutes and resuspended in Nuclei FACS Buffer (1x DPBS, 0.5% BSA, 0.5U/mL SUPERase•In, 0.5U/mL RNase Inhibitor, 2x EDTA-free Halt protease inhibitor cocktail). NeuN+ nuclei were collected with a BD FACSAria cell sorter, inspected with a Countess automated cell counter, and loaded into a 10x Genomics Chromium controller. RNA libraries were prepared with Chromium Single Cell 3' Reagent Kits (V2) according to the manufacturer's instruction